Modified integrin polypeptides, modified integrin polypeptide dimers, and uses thereof

ABSTRACT

Described herein are modified integrin α and/or β headpiece polypeptides, and crystallizable integrin polypeptide dimers comprising a modified integrin α and/or β headpiece polypeptide and a disulfide bond linking the two integrin headpiece polypeptide subunits. Methods for using the modified integrin α and/or β headpiece polypeptides and the integrin polypeptide dimers are also provided herein. For example, methods for characterizing integrin-ligand interaction and identifying integrin ligands are also provided herein. In some embodiments, the identified integrin ligands can be used as inhibitors of integrins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 16/296,350 filed Mar. 8, 2019, which is adivisional under 35 U.S.C. § 121 of U.S. application Ser. No. 15/424,260filed Feb. 3, 2017 and now U.S. patent Ser. No. 10/273,283, which is acontinuation-in-part application of PCT Application No. PCT/US15/44093filed Aug. 6, 2015, which claims benefit under 35 U.S.C. § 119(e) of theU.S. Provisional Application No. 62/033,699 filed Aug. 6, 2014, thecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 3, 2017, isnamed 701039-080812-CIP.txt and is 869,592 bytes in size.

TECHNICAL FIELD

The present invention relates to modified integrin polypeptides,crystallizable dimers comprising a modified integrin polypeptide, andmethods of using the same. Methods for characterizing integrin-ligandinteraction and identifying integrin ligands are also provided herein.The identified integrin ligands can be used as inhibitors of integrins.

BACKGROUND

Integrins are α/β heterodimers and act as cell surface receptors thatmediate cell to cell, or cell to extracellular matrix adhesion.Integrins connect diverse extracellular ligands to the cytoskeleton andregulate cell growth and differentiation. Hynes (2002) Cell 110: 673.The primary function of most of the twenty-four vertebrate integrins isto mediate cell adhesion and migration; in contrast, integrins α_(v)β₆and α_(v)β₈ are specialized to activate TGF-β1 and β3. Munger et al.(1999) Cell 96: 319; and Mu et al. (2002) J. Cell Biol. 157: 493. Thesimilarity in phenotypes of mice deficient in TGF-β1 (Shull et al.(1992) Nature 359: 693), integrin α_(V)β₆ (Munger et al. (1999) Cell 96:319) and α_(V)β₈ (Mu et al. (2002) J. Cell Biol. 157: 493), and mice inwhich RGE in pro-TGF-β1 replaces RGD (Yang et al. (2007) J. Cell Biol.176: 787), shows the importance of the RGD motif and integrins α_(V)β₆and α_(V)β₈ in TGF-β1 activation in vivo. How integrins α_(V)β₆ andα_(V)β₈ achieve specificity, and how integrin β-subunits in generalcontribute to ligand specificity remains unclear. Little is known beyondmutational evidence for the importance of a disulfide-bonded loop (theβ2-β3 loop) in the βI domain (Takagi et al. (1997) J. Biol. Chem. 272:19794), and invariant binding of the metal ion dependent adhesion site(MIDAS) to an acidic residue present in all integrin ligands (Xiong etal. (2002) Science 296: 151; Xiao et al. (2004) Nature 432: 59; Nagae etal. (2012) J. Cell Biol. 197: 131; and Sen et al. (2013) J. Cell Biol.203: 629). The issue of how the β-subunit contributes specificity isparticularly acute for the five RGD-recognizing integrins that containthe α_(V) subunit and only differ in having the β₁, β₃, β₅, β₆, or β₈subunit.

Further, integrins represent a target for treatment of various diseasesor disorders, including, e.g., inflammatory diseases, anti-angiogenictherapy, and anti-thrombotic therapy, among others. Thus, screening forand identifying new small molecules that bind to the integrin ligandbinding site and block interaction with its natural ligand canfacilitate drug discovery process.

However, the fact that functional integrins are dimeric molecules makesstudy and screening of molecules that affect their function andinteractions challenging. Therefore, there remains a need for methods tofacilitate methods and assays for screening and characterizingintegrin-ligand interaction, and thus identifying integrin ligands,e.g., for therapeutic treatment.

SUMMARY

Integrins are generally non-covalently linked heterodimers of α and βsubunits. Thus, the integrin heterodimers can reversibly dissociate,which in turn makes the process of characterizing an integrin-bindinginteraction and/or identifying a ligand that binds to the integrinheterodimer more difficult or the outcomes less reliable. The inventorshave identified modifications that allow covalent linking of integrinsthus allowing a more reliable use of them in drug screening assays.

Embodiments of various aspects described herein are, at least in part,based on development of cross-linkable integrin α and β polypeptidesubunits, which can form covalently-linked α/β heterdimers through atleast one disulfide bond, but the function of which closely mimics or isidentical to naturally occurring integrin dimers. These covalentlylinked heterodimers allow the inventors also to provide assays andmethods for screening for agents, including small molecules, peptidesand other kinds of molecules, for their potential to alter the functionof integrins.

The inventors modified the integrin α and β polypeptide subunits, forexample, by introducing at least one cysteine residue into an integrin αheadpiece polypeptide and an integrin β headpiece polypeptide. Forexample, the inventors have modified integrin α_(V) headpiece and β₆headpiece polypeptides to introduce a disulfide bond that canconvalently link the two headpiece polypeptides together.

Based on the crystal structure of integrin α_(V)β₆ heterodimer, theinventors chose to modify residues in domain(s) that are distal from theligand-binding sites, e.g., residue(s) in the α_(v) subunit β-propellerdomain and in the β₆ subunit βI domain. These sites were modified tointroduce a cysteine substitution. They discovered that the resultingdimers were surprisingly stable but also retained the functionality ofthe natural integrins. The inventors further added an extra glycineresidue into the integrin α, headpiece polypeptide at specificlocations. This modification surprisingly further improvedcrystallization and/or expression of an integrin α_(V)β₆ heterodimerupon binding with a test agent. Thus, the inventors showed thatcovalently-linked integrin dimers, such as the covalently-linkedintegrin α_(V)β₆ heterodimers, can be used to facilitate discovery ofnovel ligands for integrin heterodimers, such as α_(V)β₆ heterodimers.

Further, based on the covalently-linked integrin heterodimer, such asα_(V)β₆ headpiece heterodimer, the inventors identified a novelhydrophobic binding pocket which is a novel target site for theseintegrin headpieces. This particular hydrophobic pocket can be used notonly in vitro, but also in silico for facilitating discovery of potentligands or inhibitors that can modify, i.e., agonize or antagonize,natural integrin heterodimer function.

In addition, the inventors have introduced cysteine(s) in the integrinβ₃ and β₈ subunits at the same position structurally as in the integrinβ₆ subunits, and thus generated covalently-linked integrin α_(V)β₃ andα_(V)β₈ heterodimers, which can be crystallized to form crystalstructures with a much higher resolution (e.g., less than 3 Å or lessthan 2 Å or less than 1 Å).

Generally, a full integrin headpiece dimer is a 6-domain structure, as awild-type integrin α headpiece polypeptide includes β-propeller domainand a thigh domain, while a wild-type integrin β headpiece polypeptideincludes a βI domain, a hybrid domain, a PSI (plexin, semaphoring, andintegrin) domain, and an I-EGF-1 domain. Here, the inventors havesurprisingly discovered that a 3-domain integrin fragment of the αβheadpiece dimer, which contains only the α β-propeller and thigh domainsand the ββI domain, and is crosslinked using a disulfide bond asdescribed herein, can be generated with good expression. In oneembodiment, the inventors have created a functional 3-domaindisulfide-linked integrin fragment of the α_(V)β₆ headpiece dimer thatis capable of binding a ligand as the corresponding full headpiece. Suchan integrin fragment has never been previously made in good yield, butthe inventors was successfully able to express such a 3-domain integrinfragment by introducing a disulfide bond to crosslink the α headpiecefragment and the β headpiece fragment. Thus, not only have the inventorsgenerated disulfide-linked full headpiece of integrin dimers, but theyhave also successfully generated functional fragments of integrinheadpiece dimers (e.g., 3-domain structure) that can bind a ligand asthe corresponding full headpiece dimer. Accordingly, fragments ofmodified polypeptide dimers, e.g., 3-domain structure, 4-domainstructure, and 5-domain structure, are also described herein. Themulti-domain structures can be formed from any combination of the 6domains derived from the integrin α and β headpieces. In someembodiments, the β-domain integrin polypeptide dimer can comprise,essentially consist of, or consist of a β-propeller domain and a thighdomain of a modified integrin α headpiece incorporated with at least oneor more cysteine substitutions as described herein (e.g., modifiedintegrin α_(V) headpiece polypeptide described herein), and a βI domainof a modified integrin β headpiece incorporated with at least one ormore cysteine substitutions as described herein (e.g., modified integrinβ₆ headpiece polypeitde described herein). In some embodiments, the4-domain integrin polypeptide dimer can comprise, essentially consistof, or consist of a β-propeller domain and a thigh domain of a modifiedintegrin α headpiece incorporated with at least one or more cysteinesubstitutions as described herein (e.g., modified integrin xv headpiecepolypeptide described herein), and a βI domain and a hybrid domain of amodified integrin β headpiece incorporated with at least one or morecysteine substitutions as described herein (e.g., modified integrin β₆headpiece polypeptide described herein). In some embodiments, the5-domain integrin polypeptide dimer can comprise, essentially consistof, or consist of a β-propeller domain and a thigh domain of a modifiedintegrin α headpiece incorporated with at least one or more cysteinesubstitutions as described herein (e.g., modified integrin α_(V)headpiece polypeptide described herein), and a βI domain, a hybriddomain, and a PSI domain of a modified integrin β headpiece incorporatedwith at least one or more cysteine substitutions as described herein(e.g., modified integrin β₆ headpiece polypeptide described herein).

Accordingly, various aspects described herein provide for novelcompositions, e.g., modified integrin headpiece polypeptides, andcrystallizable integrin polypeptide dimers comprising at least onemodified integrin headpiece polypeptide, and methods of using the same.In some embodiments, the methods described herein comprise using one ormore of the compositions described herein to characterize an integrindimer-ligand interaction and/or to identify whether a test agent forms acomplex with the integrin dimer. In some embodiments, the methodsdescribed herein comprise using one or more of the compositionsdescribed herein to determine binding affinity of a test agent to anintegrin dimer. Methods for identifying a ligand, such as an inhibitoror an agonist, that bind to integrin heterodimers, such as αvβ6heterodimer are also provided herein.

In one aspect, provided herein is a modified integrin α_(V) headpiecepolypeptide. The modified integrin α_(V) headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α_(V) headpiece polypeptide (SEQ ID NO: 1) or afunctional variant thereof, with at least one Cys residue introducedthereto by one or more (e.g., at least one, at least two or more) of thefollowing modifications (a)-(e):

-   -   a. substitution of amino acid residues 399-401 (Ser-Met-Pro)        with one of the following: (i) Ser-Cys-Pro; (ii)        Gly-Cys-Pro; (iii) Ser-Cys-Gly; (iv) Gly-Cys-Gly; (v)        Ser-Gly-Cys-Pro (SEQ ID NO: 59); (vi) Ser-Cys-Gly-Pro (SEQ ID        NO: 60); (vii) Gly-Cys-Gly-Pro (SEQ ID NO: 61); and (viii)        Ser-Gly-Cys-Gly (SEQ ID NO: 62).    -   b. substitution of amino acid residues 310-311 (Gln-Glu) with        Gly-Cys;    -   c. substitution of amino acid residues 299 (Leu) and 310 (Gln)        with Cys and Gly, respectively;    -   d. substitution of amino acid residues 302-311        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln-Glu) (SEQ ID NO: 63) with        Gly-Gln-Gly-Cys (SEQ ID NO: 64); and    -   e. substitution of amino acid residue 299 (Leu) to Cys and        substitution of amino acid residues 302-310        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln) (SEQ ID NO: 65) with        Gly-Gln-Gly.

The modified integrin α_(V) headpiece polypeptide can be isolated. Themodified integrin α_(v) headpiece polypeptide can further be attached toa solid surface.

In some embodiments, the modified integrin α_(V) headpiece polypeptidecan comprise, consist essentially of, or consist of an amino acidsequence of an integrin α_(V) headpiece polypeptide (SEQ ID NO: 1) or afunctional variant thereof, with at least one Cys residue introducedthereto by substitution of amino acid residues 399-401 (Ser-Met-Pro)with Ser-Cys-Pro (modification (a)(i)). In some embodiments, themodified integrin α_(V) headpiece polypeptide can comprise, consistessentially of, or consist of an amino acid sequence of an integrinα_(V) headpiece polypeptide (SEQ ID NO:1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withGly-Cys-Pro (modification (a)(ii)). In some embodiments, the modifiedintegrin α_(V) headpiece polypeptide can comprise, consist essentiallyof, or consist of an amino acid sequence of an integrin α_(V) headpiecepolypeptide (SEQ ID NO: 1) or a functional variant thereof, with atleast one Cys residue introduced thereto by substitution of amino acidresidues 399-401 (Ser-Met-Pro) with Ser-Gly-Cys-Pro (SEQ ID NO: 59)(modification (a)(v)).

In some embodiments, the modified integrin α_(V) headpiece polypeptidecan comprise, consist essentially of, or consist of an amino acidsequence of an integrin α_(V) headpiece polypeptide (SEQ ID NO:1) or afunctional variant thereof, with at least two Cys residues introducedthereto by at least two or more of the modifications (a)-(e) asdescribed above. By way of example only, in some embodiments, at leasttwo Cys residues can be introduced into the modified integrin α_(V)headpiece polypeptide by (1) one of the modifications (a) (i)-(viii) asdescribed above; and (2) at least one of the modifications (b)-(e) asdescribed above.

In some embodiments, the modified integrin α_(V) headpiece polypeptidedescribed herein is a soluble polypeptide.

In another aspect, provided herein is a modified integrin β₆ headpiecepolypeptide. The integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consist of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by one, two, or allthree of the following modifications (f)-(h):

-   -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.

The modified integrin β₆ headpiece polypeptide can be isolated. Themodified integrin β₆ headpiece polypeptide can further be attached to asolid surface.

In some embodiments, the modified integrin β₆ headpiece polypeptide cancomprise, consist essentially of, or consist of an amino acid sequenceof an integrin β₆ headpiece polypeptide (SEQ ID NO: 2) or a functionalvariant thereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residue 270 (Ile) with Cys (modification(f)).

In some embodiments, the modified integrin β₆ headpiece polypeptide cancomprise, consist essentially of, or consist of an amino acid sequenceof an integrin β₆ headpiece polypeptide (SEQ ID NO: 2) or a functionalvariant thereof, with at least two Cys residues introduced thereto by atleast two or more of the modifications (f)-(h) as described above. Byway of example only, in some embodiments, at least two Cys residues canbe introduced into the modified integrin β₆ headpiece polypeptide by anytwo of the modifications (f)-(h) as described above.

In some embodiments, the modified integrin β₆ headpiece polypeptidedescribed herein is a soluble polypeptide.

In some aspects, provided herein are fragments of the modified integrinβ₆ headpiece polypeptides described herein. In one aspect, the integrinβ₆ headpiece polypeptide fragment comprises, consists essentially of, orconsists of a βI domain of integrin β₆ subunit with at least one Cysresidue introduced thereto by one, two, or all of the followingmodifications (f)-(h), the βI domain is defined from residues DYP toresidues ELR as shown in an amino acid sequence of SEQ ID NO: 2, or afunctional variant thereof

-   -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.

In another aspect, the integrin β₆ headpiece polypeptide fragmentcomprises, consists essentially of, or consists of a βI domain and ahybrid domain of integrin β₆ subunit, the βI domain being defined fromresidues DYP to residues ELR as shown in an amino acid sequence of SEQID NO: 2, or a functional variant thereof, while the hybrid domain beingdefined from residues ENP to residues QTE, and/or from residues SEV toresidues ECN as shown in an amino acid sequence of SEQ ID NO: 2, or afunctional variant thereof; wherein at least one least one Cys residueis introduced to the βI domain by one, two, or all of the followingmodifications (f)-(h):

-   -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.

In yet another aspect, the integrin β₆ headpiece polypeptide fragmentcomprises, consists essentially of, or consists of a βI domain, a hybriddomain, and a PSI domain of integrin β₆ subunit, the βI domain beingdefined from residues DYP to residues ELR as shown in an amino acidsequence of SEQ ID NO: 2, or a functional variant thereof, while thehybrid domain being defined from residues ENP to residues QTE, and/orfrom residues SEV to residues ECN as shown in an amino acid sequence ofSEQ ID NO: 2, or a functional variant thereof; and the PSI domain beingdefined from from residues HVQ to residues NFI as shown in an amino acidsequence of SEQ ID NO: 2; wherein at least one least one Cys residue isintroduced to the βI domain by one, two, or all of the followingmodifications (f)-(h):

-   -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.

The modified integrin β₆ headpiece polypeptide fragments of variousaspects described herein can be isolated. The modified integrin β₆headpiece polypeptide fragments of various aspects described herein canfurther be attached to a solid surface.

In some embodiments, the modified integrin β₆ headpiece polypeptidefragments of various aspects described herein are soluble polypeptides.

In another aspect, provided herein is a modified integrin β₃ headpiecepolypeptide. The integrin β₃ headpiece polypeptide comprises,essentially consist of, or consist of amino acid residues 27 to 498 ofSEQ ID NO: 5 or a functional fragment thereof (e.g., with desireddomain(s)) with at least one Cys residue introduced thereto bysubstitution of amino acid residue 293 (Gln) with Cys. The modifiedintegrin β₃ headpiece polypeptide can be isolated. The modified integrinβ₃ headpiece polypeptide can further be attached to a solid surface.

In another aspect, provided herein is a modified integrin β₈ headpiecepolypeptide. The integrin β₈ headpiece polypeptide comprises,essentially consist of, or consist of amino acid residues 43 to 498 ofSEQ ID NO: 6 or a functional fragment thereof (e.g., with desireddomain(s)) with at least one Cys residue introduced thereto bysubstitution of amino acid residue 301 (Val) with Cys. The modifiedintegrin β₈ headpiece polypeptide can be isolated. The modified integrinβ₈ headpiece polypeptide can further be attached to a solid surface.

The inventors have also introduced at least one disulfide bond tointegrin heterodimers, including, but not limited to integrin α_(V)β₆headpiece heterodimer, integrin α₅β₁ headpiece heterodimer, integrinα_(V)β₃ headpiece heterodimer, and integrin α_(V)β₈ headpieceheterodimer. Thus, modified integrin heapiece polypeptide dimerscomprising at least one of the modified integrin α headpiece polypeptideand the modified integrin β headpiece polypeptide are also providedherein. In accordance with some aspects described herein, the modifiedintegrin polypeptide dimers comprise at least one or more (e.g., atleast one, at least two, at least three or more) disulfide bonds linkingthe two integrin α and β headpiece polypeptides or functional fragmentsthereof. The modified integrin polypeptide dimer can be isolated orpurified. The modified integrin polypeptide dimer can further beattached to a solid surface.

One aspect of the modified integrin polypeptide dimers provided hereinrelates to a modified integrin polypeptide dimer comprising, consistingessentially of, or consisting of a modified integrin α headpiecepolypeptide described herein, and an integrin β polypeptide comprising,consisting essentially of, or consisting of a headpiece of the integrinsubunit, wherein the modified integrin α headpiece polypeptide and theintegrin β polypeptide are covalently linked by at least one (e.g., atleast one, at least two, at least three or more) disulfide bonds.Generally, the integrin α polypeptide can comprise, essentially of, orconsist of a β-propeller domain and a thigh domain. In some embodiments,the integrin β polypeptide can comprise, essentially consist of, orconsist of a βI domain. In some embodiments, the integrin β polypeptidecan comprise, essentially consist of, or consist of a βI domain and ahybrid domain. In some embodiments, the integrin β polypeptide cancomprise, essentially consist of, or consist of a βI domain, a hybriddomain, and a PSI domain.

The modified integrin α headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin αheadpiece polypeptide (for example, one of the integrin α headpiecepolypeptides selected from the group consisting of α₁, α₂, α₃, α₄, α₅,α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D), α_(E), α_(L), α_(M), α_(V), α_(2B), andα_(X)) or a functional variant thereof, with at least one or more (e.g.,at least one, at least two or more) Cys residues introduced thereto.

In some embodiments, the modified integrin α headpiece polypeptide is amodified integrin α_(V) headpiece polypeptide described herein. In someembodiments, the modified integrin α_(V) headpiece polypeptide cancomprise, consist essentially of, or consist of a substitution of aminoacid residues 399-401 (Ser-Met-Pro) with Ser-Cys-Pro (modification (a)(i)). In some embodiments, the modified integrin α_(V) headpiecepolypeptide can comprise, consist essentially of, or consist of asubstitution of amino acid residues 399-401 (Ser-Met-Pro) withGly-Cys-Pro (modification (a) (ii)). In some embodiments, the modifiedintegrin α_(V) headpiece polypeptide can comprise, consist essentiallyof, or consist of a substitution of amino acid residues 399-401(Ser-Met-Pro) with Ser-Gly-Cys-Pro (SEQ ID NO: 59) (modification (a)(v)).

In various embodiments, the integrin β polypeptide (comprising,consisting essentially of, or consisting of a headpiece of the integrinsubunit) covalently linked to a modified integrin α headpiecepolypeptide described herein (e.g., a modified integrin α_(V) headpiecepolypeptide described herein) can be selected from the group consistingof β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈.

Another aspect of the modified integrin polypeptide dimers providedherein relates to a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of an integrin α polypeptide(comprising, consisting essentially of, or consisting of a headpiece ofthe integrin subunit), and a modified integrin β headpiece polypeptideor a modified integrin β headpiece polypeptide fragment describedherein, wherein the integrin α polypeptide and the modified integrin βheadpiece polypeptide or the modified integrin β headpiece polypeptidefragment are covalently linked by at least one (e.g., at least one, atleast two, at least three or more) disulfide bonds.

The modified integrin β headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin βheadpiece polypeptide (for example, one of the integrin β headpiecepolypeptides selected from the group consisting of β₁, β₂, β₃, β₄, β₅,β₆, β₇, and β₈) or a functional variant thereof, with at least one ormore (e.g., at least one, at least two or more) Cys residues introducedthereto.

In some embodiments, the modified integrin β headpiece polypeptide is amodified integrin β₆ headpiece polypeptide described herein or afunctional variant thereof (e.g., a βI domain alone, or in combinationwith a hybrid domain and/or a PSI domain). In some embodiments, themodified integrin β₆ headpiece polypeptide or a functional variantthereof can comprise, consist essentially of, or consist of asubstitution of amino acid residue 270 (Ile) with Cys (modification(f)). It should be noted that numbering is based on SEQ ID NO: 2, whichis the amino acid sequence of the β₆ full headpiece. One of skill in theart can adjust the numbering of the corresponding cysteine substitution,e.g., when only a βI domain is used.

In some embodiments, the modified integrin β headpiece polypeptide is amodified integrin β₃ headpiece polypeptide described herein or afunctional variant thereof (e.g., a βI domain alone, or in combinationwith a hybrid domain and/or a PSI domain). In some embodiments, themodified integrin β₃ headpiece polypeptide or a functional variantthereof can comprise, consist essentially of, or consist of asubstitution of amino acid residue 293 (Gln) with Cys. It should benoted that numbering is based on SEQ ID NO: 5, which is the amino acidsequence of the β₃ full headpiece. One of skill in the art can adjustthe numbering of the corresponding cysteine substitution, e.g., whenonly a βI domain is used.

In some embodiments, the modified integrin β headpiece polypeptide is amodified integrin β₈ headpiece polypeptide described herein or afunctional variant thereof (e.g., a βI domain alone, or in combinationwith a hybrid domain and/or a PSI domain). In some embodiments, themodified integrin β₈ headpiece polypeptide or a functional variantthereof can comprise, consist essentially of, or consist of asubstitution of amino acid residue 301 (Val) with Cys. It should benoted that numbering is based on SEQ ID NO: 6, which is the amino acidsequence of the β₈ full headpiece. One of skill in the art can adjustthe numbering of the corresponding cysteine substitution, e.g., whenonly a βI domain is used.

In some embodiments, the integrin α polypeptide covalently linked to themodified integrin β headpiece polypeptide (e.g., a modified integrin β₆headpiece polypeptide) can be selected from the group consisting of α₁,α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D), α_(E), α_(L), α_(M),α_(V), α_(2B), and α_(X). In one embodiment, the integrin α polypeptidecovalently linked to the modified integrin β₆ headpiece polypeptide isan integrin α_(V) polypeptide comprising, consisting essentially of, orconsisting of the integrin subunit.

A further aspect of the modified integrin polypeptide dimers providedherein relates to a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of a modified integrin αheadpiece polypeptide described herein or a functional variant thereof,and a modified integrin β headpiece polypeptide described herein or afunctional variant thereof, wherein the modified integrin α headpiecepolypeptide or a functional variant thereof and the modified integrin βheadpiece polypeptide or a functional thereof are covalently linked byat least one (e.g., at least one, at least two, at least three or more)disulfide bonds.

The modified integrin α headpiece polypeptide or a functional variantthereof comprises, consists essentially of, or consists of an amino acidsequence of an integrin α headpiece polypeptide (for example, one of theintegrin α headpiece polypeptides selected from the group consisting ofα₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D), α_(E), α_(L),α_(M), α_(V), α_(2B), and α_(X)) or a functional variant thereof, withat least one or more (e.g., at least one, at least two or more) Cysresidues introduced thereto; while the modified integrin β headpiecepolypeptide comprises, consists essentially of, or consists of an aminoacid sequence of an integrin β headpiece polypeptide (for example, oneof the integrin β headpiece polypeptides selected from the groupconsisting of pi, β₂, β₃, β₄, β₅, β₆, β₇, and β₈) or a functionalvariant thereof, with at least one or more (e.g., at least one, at leasttwo or more) Cys residues introduced thereto.

In one aspect, provided herein is a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α₅ headpiece polypeptide and a modified integrin β₁ headpiecepolypeptide covalently linked together by at least one or more disulfidebond. In one embodiment, the modified integrin α₅ headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α₅ headpiece polypeptide (e.g., SEQ ID NO: 3)with at least one Cys residue introduced thereby by substitution ofamino acid residue 452 (Thr) with Cys. (SEQ ID NO: 3 includes the signalpeptide sequence at positions 1-41.) In one embodiment, the modifiedintegrin pi headpiece polypeptide comprises, consists essentially of, orconsists of one, two, three, or all domain of an integrin β₁ polypeptideheadpiece (e.g., SEQ ID NO: 4) selected from the group consisting of aPSI domain, hybrid domain, βI domain, and an EGF-1 domain, with at leastone Cys residue introduced thereby by substitution of amino acid residue295 (Leu) with Cys. (SEQ ID NO: 4 includes the signal peptide sequenceat positions 1-20.)

In one aspect, a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of a modified integrin α_(V)headpiece polypeptide described herein and a modified integrin β₃headpiece polypeptide covalently linked together by at least onedisulfide bond is also provided herein. In one embodiment, the modifiedintegrin β₃ headpiece polypeptide comprises, consists essentially of, orconsists of one, two, three, or all domain of an integrin β₃ polypeptideheadpiece (e.g., SEQ ID NO: 5) selected from the group consisting of aPSI domain, hybrid domain, βI domain, and an EGF-1 domain, with at leastone Cys residue introduced thereby by substitution of amino acid residue293 (Gln) with Cys. (SEQ ID NO: 5 includes the signal peptide sequenceat positions 1-26.)

One aspect provided herein relates to a modified integrin polypeptidedimer comprising, consisting essentially of, or consisting of a modifiedintegrin α_(V) headpiece polypeptide described herein and a modifiedintegrin β₈ headpiece polypeptide covalently linked together by at leastone disulfide bond. In one embodiment, the modified integrin β₈headpiece polypeptide comprises, consists essentially of, or consists ofone, two, three, or all domain of an integrin β₈ polypeptide headpiece(e.g., SEQ ID NO: 6) selected from the group consisting of a PSI domain,hybrid domain, βI domain, and an EGF-1 domain, with at least one Cysresidue introduced thereby by substitution of amino acid residue 301(Val) with Cys. (SEQ ID INO: 6 includes the signal peptide sequence atpositions 1-42.)

A further aspect provides a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α_(V) headpiece polypeptide described herein and a modifiedintegrin β₆ headpiece polypeptide covalently linked together by at leastone disulfide bond. The modified integrin polypeptide dimer comprises,consists essentially of, or consists of (i) an integrin α_(V) headpiecepolypeptide or a functional variant thereof, with at least one or more(e.g., at least one, at least two or more) Cys residues introducedthereto; and (ii) an integrin β₆ headpiece polypeptide or a functionalvariant thereof, with at least one or more (e.g., at least one, at leasttwo or more) Cys residues introduced thereto.

In some embodiments, the modified integrin polypeptide dimer comprises,consists essentially of, or consists of a modified integrin α_(V)headpiece polypeptide and a modified integrin β₆ headpiece polypeptidecovalently linked together by at least one or more disulfide bonds,wherein:

the modified integrin α_(V) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(V) headpiece polypeptide (SEQ ID NO: 1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withSer-Cys-Pro (modification (a)(i)); and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by substitution ofamino acid residue 270 (Ile) with Cys (modification (f)).

In some embodiments, the modified integrin polypeptide dimer comprises,consists essentially of, or consists of a modified integrin α_(V)headpiece polypeptide and a modified integrin β₆ headpiece polypeptidecovalently linked together by at least one or more disulfide bonds,wherein:

the modified integrin α_(V) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(V) headpiece polypeptide (SEQ ID NO: 1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withGly-Cys-Pro (modification (a)(ii)); and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by substitution ofamino acid residue 270 (Ile) with Cys (modification (f)).

In some embodiments, the modified integrin polypeptide dimer comprises,consists essentially of, or consists of a modified integrin α_(V)headpiece polypeptide and a modified integrin β₆ headpiece polypeptidecovalently linked together by at least one or more disulfide bonds,wherein:

the modified integrin α_(v) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(v) headpiece polypeptide (SEQ ID NO: 1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withSer-Gly-Cys-Pro (SEQ ID NO: 59) (SEQ ID NO: 59) (modification (a)(v));and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by substitution ofamino acid residue 270 (Ile) with Cys (modification (f)).

In some embodiments, the modified integrin polypeptide dimers of variousaspects described herein can be soluble polypeptides.

The modified integrin polypeptide dimers (e.g., comprising, consistingessentially of, or consisting of a modified integrin α_(V) headpiecepolypeptide described herein, and a modified integrin β₆ headpiecepolypeptide described herein) can still interact with a natural ligandas it binds to a naturally occurring integrin heterodimer, but, unlikenaturally occurring integrin heterodimers that can reversiblydissociate, the modified headpiece polypeptides described herein do notdissociate and enable formation of a crystallizable structure of anintegrin heterodimer, alone or complexed with a test ligand, viaformation of a disulfide bridge between the two integrin headpiecesubunits. Accordingly, in some aspects, the compositions as describedherein can be used to characterize an integrin-ligand interaction, e.g.,to measure the binding affinity of a test agent to an integrinheterodimer, e.g., integrin α_(V)β₆ heterodimer; and/or to identify anovel integrin ligand, e.g., in a drug discovery process.

For example, a method for determining whether a test agent forms acomplex with an integrin is provided herein. The method comprisescontacting one or more of the modified polypeptide described herein witha test agent, and detecting formation of a complex comprising themodified integrin polypeptide dimer and the test agent bound thereto.Detection of a formed complex comprising the modified integrinpolypeptide dimer and the test agent bound thereto indicates that thetest agent is capable of forming a complex with the integrin.

Various methods known in the art can be used to detect formation of acomplex comprising the modified integrin polypeptide dimer and a testagent bound thereto. By way of example only, in some embodiments, thecomplex can be detected by a detection method comprising crystallizationof the complex. In some embodiments, the test agent can further comprisea detectable label. Examples of a detectable label include, but are notlimited to, biotin, a fluorescent dye or molecule, a luminescent orbioluminescent marker, a radiolabel, an enzyme, a quantum dot, animaging agent, or any combination thereof. Methods for detecting varioustypes of the detectable labels are known in the art. For example, wherethe detectable label comprises a fluorescent molecule, signals from thefluorescent labels can be detected, e.g., by fluorescence anisotropyand/or flow cytometry.

In some embodiments, instead of directly detecting a test agent bound tothe modified integrin polypeptide dimer, binding of the test agent tothe modified integrin polypeptide dimer can also be determined by anindirect method, e.g., a competition binding assay. In a competitionbinding assay, the method can further comprise, prior to the detecting,contacting the modified integrin polypeptide dimer with a competingagent.

A competing agent is an agent capable of competing with a test agent tobind the modified integrin polypeptide dimer. Accordingly, a competingagent can be a protein, a peptide, an antibody, a nucleic acid molecule,an apatmer, a peptidomimetic, a small molecule, or any combinationsthereof. In some embodiments, the competing agent can be a competingpeptide.

Since the modified integrin polypeptide dimer is crystallizable, thebinding domain of the dimer can be readily identified using any methodsknown in the art, e.g., X-ray crystallography. Based on the bindingdomain of the dimer, one can design a competing agent that can bind tothe binding domain. An exemplary competing peptide for binding to amodified α_(V)β₆ polypeptide dimer described herein comprises an aminoacid sequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu (SEQ ID NO: 66), whereinX₁, X₂, and X₃ are each independently an amino acid molecule. Analternative competing peptide for binding to a modified α_(V)β₆polypeptide dimer described herein comprises an amino acid sequence ofX₃-Arg-Gly-Asp-Leu-X₁-X₂-Ile (SEQ ID NO: 67), wherein X₁, X₂, and X₃ areeach independently an amino acid molecule.

In some embodiments, the X₁ can be a Gly molecule or an analog thereof.

In some embodiments, the X₂ can be an Arg molecule or an analog thereof.

In some embodiments, the X₃ can be a Gly molecule or an analog thereof.

In some embodiments, a competing peptide for binding to a modifiedα_(V)β₆ polypeptide dimer described herein can comprise an amino acidsequence of Gly-Arg-Gly-Asp-Leu-Gly-Arg-Leu (SEQ ID NO: 68). In someembodiments, a competing peptide for binding to a modified α_(V)β₆polypeptide dimer described herein can comprise an amino acid sequenceof Gly-Arg-Gly-Asp-Leu-Gly-Arg-Ile (SEQ ID NO: 69).

In some embodiments, the competing agent can further comprise adetectable label. Examples of a detectable label include, but are notlimited to, biotin, a fluorescent dye or molecule, a luminescent orbioluminescent marker, a radiolabel, an enzyme, a quantum dot, animaging agent, or any combination thereof. Methods for detecting varioustypes of the detectable labels are known in the art. For example, wherethe detectable label comprises a fluorescent molecule, signals from thefluorescent labels can be detected, e.g., by fluorescence anisotropyand/or flow cytometry.

Where the competing agent comprises a detectable label, signals from thecompeting agent is detected instead of the test agent. Thus, if thesignal from the competing agent is reduced upon contacting the modifiedintegrin polypeptide dimer with the test agent, this indicates that thetest agent has a higher binding affinity than the competing agent to themodified integrin polypeptide dimer described herein.

Accordingly, yet another aspect provided herein relates to a method fordetermining binding affinity of a test agent to an integrin. The methodcomprises (i) contacting one or more modified integrin polypeptidedimers described herein with a test agent and a competing agent, whereinthe competing agent comprises a detectable label and is capable ofcompeting with the test agent to bind the modified integrin polypeptidedimer; and (ii) detecting a signal from the detectable label of thecompeting agent that forms a complex with the integrin, whereby adecrease in the detected signal relative to a signal corresponding tosaturation binding of the competing agent to the modified integrinpolypeptide dimer indicates that the test agent has a higher bindingaffinity than the competing agent to the integrin. In some embodiments,the concentrations of the test agent and the competing agent areessentially the same.

As noted above, the modified integrin polypeptide dimers describedherein can form crystal structures. Thus, the binding domain of thedimer can be readily identified using any methods known in the art,e.g., X-ray crystallography. Indeed, the inventors have identified anovel hydrophobic binding pocket of an integrin α_(V)β₆ heterodimerbased on the crystal structure thereof. Accordingly, inventors employthe information of the novel hydrophobic binding pocket to design apharmacophore model for an agent that can bind to the hydrophobicbinding pocket of the integrin α_(V)β₆ heterodimer.

In some embodiments, the pharmacophore model can be designed for ananti-α_(V)β₆ inhibitor. Therefore, provided herein is also a method ofidentifying an anti-α_(V)β₆ inhibitor. The method comprises: (a)generating on a computer a molecular representation of a pharmacophorecomprising a basic functional group, an acidic functional group forcoordination of a metal ion to a metal ion-dependent adhesion site(MIDAS) in integrin β₆ subunit, a first hydrophobic functional group,and a second hydrophobic functional group, wherein the functional groupsare arranged to satisfy the following conditions:

the distance between the first hydrophobic functional group (H1) and thesecond hydrophobic functional group (H2) is about 7-8 Å; the distancebetween the second hydrophobic functional group (H2) and the basicfunctional group (B) is about 8-9 Å; the distance between the basicfunctional group (B) and the acidic functional group (A) is about 15-16Å; the distance between the first hydrophobic functional group (H1) andthe acidic functional group (A) is about 14.5-15.5 Å; and the distancebetween the second hydrophobic functional group (H2) and the acidicfunctional group (A) is about 19-20 Å; and

the angle formed by H1-A-B is about 20°-24°; the angle formed by H1-A-H2is about 17°-21°; the angle formed by H2-A-B is about 26°-30°; the angleformed by A-B-Hi is about 68°-72°; the angle formed by A-B-H2 is about96°-100°; and the angle formed by H1-B-H2 is about 49°-53°; (b)generating on a computer atomic coordinates of an α_(V)β₆ integrinprotein or a portion thereof having at least a hydrophobic bindingpocket in β₆ subunit; and (c) determining on a computer likelihood ofthe molecular representation interacting with one or more residues ofthe computer-generated α_(V)β₆ integrin protein or a portion thereof,thereby identifying a candidate anti-α_(V)β₆ inhibitor.

For example, one can use the following: the distance between the firsthydrophobic functional group and the second hydrophobic functional groupis about 7.403 Å; the distance between the second hydrophobic functionalgroup and the basic functional group is about 8.462 Å; the distancebetween the basic functional group and the acidic functional group isabout 15.639 Å; the distance between the first hydrophobic functionalgroup and the acidic functional group is about 15.005 Å; and thedistance between the second hydrophobic functional group and the acidicfunctional group is about 19.553 Å; and the angle formed by H1-A-B isabout 22.4°; the angle formed by H1-A-H2 is about 19.4°; the angleformed by H2-A-B is about 28.7°; the angle formed by A-B-H1 is about70.7°; the angle formed by A-B-H2 is about 98.1°; and the angle formedby H1-B-H2 is about 51.1°.

In some embodiments, the first and second hydrophobic functional groupscan each independently have an aromatic ring (aryl) or linear moiety.

In another aspect, provided herein is a method of identifying ananti-α_(V)β₆ inhibitor. The method comprises or consists of or consistsessentially of: (a) providing, on a computer, a three-dimensionalcrystalline structure of α_(V)β₆ integrin protein or a portion thereofcharacterized by atomic structure coordinates (e.g., as described inTable 6 of, PCT Application No: PCT/US15/44093 filed Aug. 6, 2015) or athree-dimensional structure that exhibits a root-mean-square difference(rmsd) in α-carbon positions of less than 2.0 Å (or less than 1.0 Å)with the atomic structure coordinates (e.g., as described in Table 6 ofPCT Application No: PCT/US15/44093 filed Aug. 6, 2015); (b) docking onthe computer a first molecular entity in a first part of a bindingpocket of the integrin β₆ headpiece polypeptide having an amino acidsequence of SEQ ID NO: 2, to determine the binding association betweenthe first molecular entity and the first part of the binding pocket,wherein the binding pocket comprises amino acid residues Ala-217,Asn-218, Pro-179, Cys-180, Ile-183, Ala-126, and Tyr-185; (c) docking onthe computer a second molecular entity in a second part of the bindingpocket, to determine the binding association between the secondmolecular entity and the second part of the binding pocket; (d)repeating steps (b) to (c) with a different first and second molecularentity; (e) selecting a first and a second chemical entity based on thequantified binding associations; and (f) generating on the computer apharmacophore model by assembling the selected first and secondmolecular entity into a molecular representation that interacts with thebinding pocket.

In some embodiments of this aspect and other aspects described herein,the method can further comprise contacting the candidate anti-α_(V)β₆inhibitor (based on the pharmacophore model) with a α_(V)β₆ integrinprotein to determine the ability of the candidate anti-α_(V)β₆ integrininhibitor to bind the α_(V)β₆ integrin protein.

In a further aspect, provided herein is a crystalline compositioncomprising a ligand-binding headpiece of integrin α_(V) β₆, wherein thecrystalline composition is characterized with space group C2, and hasunit cell parameters a=184.5±3 Å, b=168.3±3 Å, c=101.8±3 Å, α=β=90°, andγ=98.2°±3°.

In some embodiments, the crystalline composition can further comprise aligand. Accordingly, a crystalline composition comprising aligand-binding headpiece of integrin α_(V)β₆ and a ligand is alsoprovided herein. The crystalline composition is characterized with spacegroup C2, and has unit cell parameters a=184.4±3 Å, b=170.0±3 Å,c=102.4±3 Å, α=β=90°, and γ=98.7°±3°.

In some embodiments, the ligand-binding headpiece of integrin α_(V)β₆can comprise a modified integrin α_(V) headpiece polypeptide describedherein, and a modified integrin β₆ headpiece polypeptide describedherein.

In some embodiments, the crystalline composition has a binding pocket inthe modified integrin β₆ headpiece polypeptide, wherein the bindingpocket comprises amino acid residues Ala-217, Asn-218, Pro-179, Cys-180,Ile-183, Ala-126, and Tyr-185.

In some embodiments, the ligand-binding headpiece of integrin β₆ can bedescribed by its atomic structure coordinates (e.g., described in Table6 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015, or astructure that exhibits a root-mean-square difference (rmsd) in α-carbonpositions of less than 2.0 Å (or less than 1.0 Å) with the atomicstructure coordinates (e.g. described in Table 6.of PCT Application No:PCT/US15/44093 filed Aug. 6, 2015.

In some embodiments, the crystalline composition can be formed from acrystallization solution buffered between pH 6-8 at a temperature ofabout 20° C. or room temperature and having an ionic strength of about800-900 mM.

By introducing at least one disulfide bond to an integrin dimer, theinteraction between the integrin α subunit and the integrin β subunit,unlike the wild-type integrin dimer, is irreversible, and thus crystalstructure of the disulfide-linked integrin dimer can be formed with ahigh resolution, which can then used for various applications, e.g.,pharmacophore modeling, and/or drug screening. Accordingly, a method ofidentifying an anti-α_(V)β₃ inhibitor is also provided herein. Themethod comprises: (a) providing, on a computer, a three-dimensionalcrystalline structure of α_(V)β₃ integrin protein or a portion thereofcharacterized by atomic structure coordinates (e.g., as described inTable 8 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015 or athree-dimensional structure that exhibits a root-mean-square difference(rmsd) in α-carbon positions of less than 2.5 Å (or less than 2.0 Å, orless than 1.0 Å) with the atomic structure coordinates (e.g., describedin Table 8 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015);(b) docking on the computer a first molecular entity in a first part ofa binding pocket of the integrin β₃ headpiece polypeptide having anamino acid sequence of SEQ ID NO: 5 or a fragment thereof (with desireddomain(s)), to determine the binding association between the firstmolecular entity and the first part of the binding pocket; (c) dockingon the computer a second molecular entity in a second part of thebinding pocket, to determine the binding association between the secondmolecular entity and the second part of the binding pocket; (d)repeating steps (b) to (c) with a different first and second molecularentity; (e) selecting a first and a second chemical entity based on thequantified binding associations; and (f) generating on the computer apharmacophore model by assembling the selected first and secondmolecular entity into a molecular representation that interacts with thebinding pocket.

In some embodiments, the method can further comprise contacting thecandidate anti-α_(V)β₃ inhibitor (based on the pharmacophore model) withan α_(V)β₃ integrin protein to determine the ability of the candidateanti-α_(V)β₃ integrin inhibitor to bind the α_(V)β₃ integrin protein.

A crystalline composition comprising a ligand-binding headpiece ofintegrin α_(V)β₃ is also provided herein. The crystalline composition ischaracterized with space group P22₁2₁, and has unit cell parametersa=87±2 Å, b=124±2 Å, c=165±2 Å, a=P=90°, and γ=90°±3°. In someembodiments, the ligand-binding headpiece of integrin α_(V)β₃ cancomprise a modified integrin α_(V) headpiece polypeptide describedherein, and a modified integrin β₃ headpiece polypeptide describedherein. In some embodiments, the ligand-binding headpiece of integrinα_(V)β₃ can be described by its atomic structure coordinates (e.g., asdescribed in Table 8 of PCT Application No: PCT/US15/44093 filed Aug. 6,2015), or a structure that exhibits a root-mean-square difference (rmsd)in α-carbon positions of less than 2.5 Å (or less than 2.0 Å, or lessthan 1.0 Å) with the atomic structure coordinates (e.g., as described inTable 8 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015). Insome embodiments, the crystal composition can be formed from acrystallization solution buffered between pH 6-8 at a temperature ofabout 20° C. and having an ionic strength of about 800-900 mM.

Another aspect provided herein relates to a crystalline compositioncomprising a ligand-binding headpiece of integrin α_(V)β₈. Theligand-binding headpiece of integrin α_(V)β₈ is characterized with spacegroup P1, and has unit cell parameters a=153±3 Å, b=55±3 Å, c=181±3 Å,α=β=90°, and γ=110°±3°. In some embodiments, the ligand-bindingheadpiece of integrin α_(V)β₈ can comprise a modified integrin α_(V)headpiece polypeptide described herein, and a modified integrin β₈headpiece polypeptide described herein. In some embodiments, the crystalcomposition can be formed from a crystallization solution bufferedbetween pH 6-8 at a temperature of about 20° C. and having an ionicstrength of about 800-900 mM.

The crystalline composition can be used for various applications,including, e.g., drug screening or pharmacophore modeling. Thus, amethod of identifying an anti-α_(V)β₈ inhibitor is also provided herein.The method comprises: (a) providing, on a computer, a three-dimensionalcrystalline structure of α_(V)β₈ integrin protein or a portion thereofderived from the crystalline composition comprising a ligand-bindingheadpiece of integrin α_(V)β₈ described herein; (b) docking on thecomputer a first molecular entity in a first part of a binding pocket ofthe integrin β₈ headpiece polypeptide having an amino acid sequence ofSEQ ID NO: 6 or a fragment thereof (e.g., with desired domain(s)), todetermine the binding association between the first molecular entity andthe first part of the binding pocket; (c) docking on the computer asecond molecular entity in a second part of the binding pocket, todetermine the binding association between the second molecular entityand the second part of the binding pocket; (d) repeating steps (b) to(c) with a different first and second molecular entity; (e) selecting afirst and a second chemical entity based on the quantified bindingassociations; and (f) generating on the computer a pharmacophore modelby assembling the selected first and second molecular entity into amolecular representation that interacts with the binding pocket. In someembodiments, the method can further comprise contacting the candidateanti-α_(V)β₈ inhibitor (based on the pharmacophore model) with anα_(V)β₈ integrin protein to determine the ability of the candidateanti-α_(V)β₈ integrin inhibitor to bind the α_(V)β₈ integrin protein.

Kits comprising at least one of the modified integrin headpiecepolypeptides or fragments thereof (e.g., but not limited to the modifiedintegrin α_(V) headpiece polypeptides described herein, and/or themodified integrin β₆ headpiece polypeptides described herein) and/or atleast one of the modified integrin polypeptide dimers are also providedherein.

In some embodiments, the modified integrin headpiece polypeptides and/orthe modified integrin polypeptide dimers included in the kit can beattached to a solid surface.

In some embodiments, the kit can further comprise at least one reagentto perform the methods described herein. For example, in one embodiment,at least one competing agent as described herein can be included in thekit.

Polynucleotides encoding the modified integrin headpiece polypeptides(e.g., the modified integrin α_(V) headpiece polypeptides describedherein, and the modified integrin β₆ headpiece polypeptides describedherein) are also encompassed within the scope of the inventionsdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show activation and binding of pro-TGF-β by wild-type (WT)and mutant α_(V) integrins. (FIG. 1A) The indicated 293T transfectantswere assayed for TGF-β1 activation using mink lung luciferase reportercells. (FIG. 1B) Saturation binding of FITC-pro-TGF-β1 to 293Ttransfectants as % mean fluorescence intensity (MFI) of α_(v) P2W7 mAbbinding. (FIG. 1C) Binding of peptides to α_(v)β₆ headpiece measured byfluorescence anisotropy (peptides disclosed as SEQ ID NOS 80-81, 75, and72, respectively, in order of appearance). (FIG. 1D) Binding of peptidesto α_(V)β₃ headpiece measured by fluorescence anisotropy. Data showmean±SEM of triplicate samples.

FIGS. 2A-2H show crystal structures and comparisons of α_(V)β₆headpiece. (FIG. 2A) Overall ribbon diagram of α_(V)β₆ headpiece withpro-TGF-β3 peptide. (FIG. 2B) Conformational change of the βI α2-α3 loopin absence and presence of pro-TGF-β₃. Carbons are shown. Metals arewhite or grey spheres. (FIG. 2C) pH-dependence of binding affinity.Binding of FITC-pro-TGF-β3 peptide was measured using fluorescenceanisotropy. (FIG. 2D) Ligand binding of α_(V)β₆ to pro-TGF-β₃ peptide.Carbons are shown. (FIG. 2E) Ligand binding of α_(V) β₃ to cilengitideas described in Xiong et al., Science 296 (2002) 51-155. Carbons areshown. (FIGS. 2F and 2G) Key residues that contribute to packing betweenSDL1, 2, and 3 in β₆ (FIG. 2F) and β₃ (FIG. 2G). Van der Waals surfacesaround interacting sidechains are shown as dots. (FIG. 2H) Phylogenetictree for integrin β subunit SDL sequences as described in Huhtala etal., Matrix Biol. 24 (2005) 83. Ligand contacting residues in SDL1 andSDL3 in X₁ positions are highlighted in a darker shade. Residues thatform packing interactions of SDL1 and 3 with SDL2 in X₂ position arehighlighted in medium shade. Cysteines forming disulfides arehighlighted in a lighter shade. Residues that coordinate metals areasterisked and indicated in the figure. Metal ion-dependent adhesionsite (MIDAS), adjacent to MIDAS (ADMIDAS); synergistic metal bindingsite (SyMBS). FIG. 2H discloses SEQ ID NOS 82-105, respectively, inorder of appearance.

FIGS. 3A-3D show experimental data on ligands of αvβ6. (FIG. 3A) RGDsequences from pro-TGF-β and VP1 protein from foot-and-mouth diseasevirus (SEQ ID NOS 106-107, 78, and 108, respectively, in order ofappearance). (FIG. 3B) Competitive binding affinities of TGF-β3 peptidetruncations (SEQ ID NOS 78 and 109-114, respectively, in order ofappearance). Fluorescence anisotropy data are mean±SEM of triplicatesamples scaled logarithmically. (FIG. 3C) Western blots of proTGF-β1secreted by the indicated 293T transfectants using antibody to theprodomain as described in Wang et al., Mol BIol. Cell 23 (2012) 1129.(FIG. 3D) TGF-β bioassay of proTGF-β1 and its double proline mutant.

FIG. 4 shows a computer-generated docking study snapshot in whichdifferent molecular entities or functional groups of an exemplarypharmacophore model are docked into various parts of binding pocket ofthe crystal structure of integrin α_(V)β₆ headpiece. Distances betweenthe functional groups are labeled in the figure. H11 and H12 are thehydrophobic group, which can be aromatic or linear. β₂ and β₁₉ are thenegative charged groups. N17 is a positive charged group when metal ionbinds at integrin MIDAS, but N17 can also be a positive charged groupwhen metal ion is absent at integrin MIDAS to replace the metal ion.There are four functional groups, and the ∠H11P19N17=22.4°,∠H11P19H12=19.4°, ∠H12P19N17=28.7°, ∠P19N17H11=70.7°, ∠P19N17H12=98.1°,∠H11N17H12=51.1°.

FIG. 5 shows reducing and non-reducing SDS-PAGE of modified integrinα_(V)β₃, α_(V)β₆, and α_(V)β₈ headpiece dimer. NR, M, R stand forNon-reducing, Marker, and Reducing.

FIG. 6 shows reducing and non-reducing SDS-PAGE of modified integrinα_(V)β₆ 3-domain fragment dimer. NR, M, R stand for Non-reducing,Marker, and Reducing.

FIGS. 7A-7B are graphs showing competitive binding affinities ofindicated inhibitors to modified integrin α_(V)β₆ (FIG. 7A) and α_(V)β₈(FIG. 7B) headpiece dimer. Fluorescence anisotropy data are mean±SEM oftriplicate samples scaled logarithmically.

FIG. 8 shows an amino acid sequence of an integrin β₆ subunit. The PSIdomain is highlighted in bold. The hybrid domain is highlighted in grey.The βI domain is highlighted in black. The EGF-1 domain is underlined.

FIG. 9 shows an amino acid sequence of an integrin β₃ subunit. Thesignal sequence is indicated in a line box. The PSI domain ishighlighted in bold. The hybrid domain is highlighted in grey. The βIdomain is highlighted in black. The EGF-1 domain is underlined.

FIG. 10 shows an amino acid sequence of an integrin β₈ subunit. Thesignal sequence is indicated in a line box. The PSI domain ishighlighted in bold. The hybrid domain is highlighted in grey. The βIdomain is highlighted in black. The EGF-1 domain is underlined.

DETAILED DESCRIPTION

As integrins are generally non-covalently linked heterodimers of α and βsubunits, the integrin heterodimers can reversibly dissociate into α andβ subunits. Therefore, characterizing an integrin-binding interactionand/or identifying a ligand that binds to the integrin heterodimer canbe difficult. The inventors have developed cross-linkable integrin α andβ polypeptide subunits, which can form covalently-linked α/β heterdimersthrough at least one disulfide bond.

For example, the inventors have modified the integrin α and β headpiecepolypeptide subunits by introducing at least one or more cysteineresidues into one or both of the α and β headpiece polypeptide subunits.For example, the inventors have modified integrin α_(V) and β₆ headpiecepolypeptide subunits to introduce a disulfide bond that can covalentlylink the two subunits together. Aspects of the invention can also beapplied to other integrins that have homologous sequences or similarstructures. The inventors utilized the crystal instructure of integrinαvβ6 heterodimer to select, one or more residues at specific locationsin domain(s) that are distal from the ligand-binding sites, e.g.,residue(s) in the α_(V) subunit β-propeller domain and in the β₆ subunitβI-domain, and modified them to introduce a cysteine substitution. Insome embodiments, the inventors further added an extra glycine residueinto the integrin α_(V) headpiece polypeptide at specific locations.Such modifications resulted in ability, e.g., to further improvecrystallization and/or expression of an integrin heterodimer, such asα_(V)β₆ heterodimer, without or upon binding with a test agent.

Thus, in one aspect, the covalently-linked integrin heterodimers, suchas integrin α_(V)β₆ heterodimers can be used to facilitate discovery ofnovel ligands for integrin α_(V)β₆ heterodimers. Further, based on thecovalently-linked integrin α_(V)β₆ heterodimer, the inventors haveidentified a novel hydrophobic binding pocket as a target site in theheadpiece for these integrins, thus facilitating discovery of potentligands or inhibitors against these integrins.

In addition, the inventors have introduced cysteine(s) in integrin β₃and β₈ subunits at the same position structurally as in the integrin β₆subunit, and thus generated covalently-linked integrin α_(V)β₃ andα_(V)β₈ heterodimers, which can be crystallized to form more stablecrystal structures with a much higher resolution (e.g., less than 3 Å orless than 2 Å or less than 1 Å).

Generally, a full integrin αβ headpiece dimer is a 6-domain structure,as a wild-type integrin α headpiece polypeptide includes β-propellerdomain and a thigh domain, while a wild-type integrin β headpiecepolypeptide includes a βI domain, a hybrid domain, a PSI (plexin,semaphoring, and integrin) domain, and an I-EGF-1 (or EGF-1) domain.Here, the inventors have surprisingly discovered that a 3-domainintegrin fragment of the αβ headpiece dimer, which contains only the αβ-propeller and thigh domains and the β βI domain, and is crosslinkedusing a disulfide bond as described herein, can be generated with goodexpression. In one embodiment, the inventors have created a functional3-domain disulfide-linked integrin fragment of the α_(V)β₆ headpiecedimer that is capable of binding a ligand as the corresponding fullheadpiece. Such an integrin fragment has never been previously made ingood yield, but the inventors were successfully able to express such a3-domain integrin fragment by introducing a disulfide bond to crosslink(covalently-link) the α headpiece and the β headpiece fragment. Thus,not only have the inventors generated disulfide-linked full headpiece ofintegrin dimers, but they have also successfully generated functionalfragments of integrin headpiece dimers (e.g., 3-domain structure) thatcan bind a ligand as the corresponding full headpiece dimer.Accordingly, fragments of modified integrin αβ polypeptide dimers, e.g.,3-domain structure, 4-domain structure, and 5-domain structure, are alsodescribed herein. The multi-domain structures can be formed from anycombination of the 6 domains derived from the integrin α and βheadpieces. In some embodiments, the 3-domain integrin polypeptide dimercan comprise, essentially consist of, or consist of a β-propeller domainand a thigh domain of a modified integrin α headpiece incorporated withat least one or more cysteine substitutions as described herein (e.g.,modified integrin α_(V) headpiece polypeptide described herein), and aβI domain of a modified integrin β headpiece incorporated with at leastone or more cysteine substitutions as described herein (e.g., modifiedintegrin β₆ headpiece polypeptide described herein). In someembodiments, the 4-domain integrin polypeptide dimer can comprise,essentially consist of, or consist of a β-propeller domain and a thighdomain of a modified integrin α headpiece incorporated with at least oneor more cysteine substitutions as described herein (e.g., modifiedintegrin xv headpiece polypeptide described herein), and a βI domain anda hybrid domain of a modified integrin β headpiece incorporated with atleast one or more cysteine substitutions as described herein (e.g.,modified integrin β₆ headpiece polypeptide described herein). In someembodiments, the 5-domain integrin polypeptide dimer can comprise,essentially consist of, or consist of a β-propeller domain and a thighdomain of a modified integrin α headpiece incorporated with at least oneor more cysteine substitutions as described herein (e.g., modifiedintegrin α_(V) headpiece polypeptide described herein), and a βI domain,a hybrid domain, and a PSI domain of a modified integrin β headpieceincorporated with at least one or more cysteine substitutions asdescribed herein (e.g., modified integrin β₆ headpiece polypeptidedescribed herein).

Accordingly, various aspects described herein provide for compositions(e.g., modified integrin headpiece polypeptides and functionalvariants/fragments thereof, and crystallizable integrin polypeptidedimers comprising at least one modified integrin headpiece polypeptide)and methods of using the same. In some embodiments, the methodsdescribed herein comprise using one or more of the compositionsdescribed herein to characterize an integrin-ligand interaction and/orto identify whether a test agent forms a complex with the integrin. Insome embodiments, the methods described herein comprise using one ormore of the compositions described herein to determine binding affinityof a test agent to an integrin. Methods for identifying a ligand thatbinds to integrin αvβ6 heterodimer (e.g., an anti-αvβ6 inhibitor) arealso provided herein.

Modified Integrin α Headpiece Polypeptide (e.g., Modified Integrin α_(v)Headpiece Polypeptides)

One aspect provided herein relates to a modified integrin α headpiecepolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence of an integrin α headpiece polypeptide or afunctional variant thereof, with at least one or more (e.g., at leastone, at least two, at least three or more) Cys residues introduced into.

In some embodiments, Cys residue(s) can be introduced into domain(s) ofan integrin α headpiece polypeptide that are distal from aligand-binding site. The modified integrin α headpiece polypeptide cansubstantially retain the functionality (e.g., ligand-binding capability)of the naturally occurring integrin α headpiece polypeptide (e.g.,retain at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 99% or more and up to 100%), while having theability to form a disulfide bond with an integrin β polypeptide or amodified integrin β headpiece polypeptide described herein.

As used herein, the term “distal from a ligand-binding site” refers toany amino acid residue or domain that does not participate in ligandbinding such that modification(s) to the amino acid residue or domaindoes not substantially impair a ligand binding to the ligand-bindingsite.

As used herein, the term “ligand-binding site” refers to a particularregion or regions of a protein or polypeptide to which a ligand binds toform a complex with the protein or polypeptide.

A region can comprise one or more individual amino acid residues. Inaccordance with various aspects described herein, the ligand-bindingsite is generally present in an integrin headpiece, which is furtherdescribed below.

The term “ligand” is generally meant to refer to a small molecule havingaffinity for a target molecule, e.g., a protein molecule. Generally, aligand can preferentially bind to a target molecule at one or moreparticular sites.

A Cys residue can be introduced into one or more domain(s) of anintegrin α headpiece polypeptide by any methods of modifying amino acidsknown in the art, including, e.g., substitution, deletion and/oraddition of an amino acid or amino acid analog. In some embodiments, atleast one or more Cys residue or an analog thereof can be introducedinto one or more domain(s) of the integrin α headpiece polypeptide bysubstitution of an amino acid residue, and/or addition of a Cys residueor an analog thereof. In some embodiments, while introducing one or moreCys residue into at least one domain of the integrin α headpiecepolypeptide, one or more other amino acid residues present in theintegrin α headpiece polypeptide can also be deleted.

As used herein, the term “analog” when used in reference to an aminoacid residue or molecule refers to a molecule that retains the samestructure or characteristic (e.g., size, charges and/or interactions) asa parent amino acid residue or molecule. In some embodiments, the analogcan include a non-proteinogenic amino acid derived from a proteinogenicamino acid.

As used herein, the term “integrin α headpiece polypeptide” refers to apolypeptide that is similar or identical to the sequence of wild-typeintegrin α polypeptide having at least the headpiece thereof Δn integrinα headpiece polypeptide generally includes β-propeller domain and/or athigh domain, and confers ligand-binding capability upon binding with anappropriate integrin β headpiece polypeptide. An integrin α headpiecepolypeptide can be derived from the headpiece of any integrin αpolypeptide, including, for example, α₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉,α₁₀, α₁₁, α_(D), α_(E), α_(L), α_(M), α_(V), α_(2B), and α_(X). In someembodiments, the term “integrin α headpiece polypeptide” refers to apolypeptide having an amino acid sequence that is at least 70% or more(including at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100%) identical to that of awild-type integrin α polypeptide having at least the headpiece thereof(including, e.g., β-propeller and/or thigh domains), and is capable ofinteracting with an integrin β subunit (e.g., a headpiece) to form aheterodimer and retaining (e.g., at least 70% or more of) the biologicalactivity of the wild-type heterodimer, including, but not limited to,ligand binding, cellular adhesion, migration, invasion, differentiation,proliferation, apoptosis, and/or gene expression. In some embodiments,the term “integrin α headpiece polypeptide” refers to a polypeptide thatis similar or identical to the sequence of a wild-type integrin αheadpiece polypeptide (including, e.g., β-propeller and/or thighdomains). In some embodiments, the integrin α headpiece polypeptide canfurther include other domain(s) of a wild-type integrin α polypeptidesuch as calf-1 and/or calf-2 domains that form the lower legs of thewild-type integrin α polypeptide. In some embodiments, an integrin αheadpiece polypeptide refers to a full-length wild-type integrin αheadpiece polypeptide sequence. In some embodiments, an integrin αheadpiece polypeptide refers to a functional domain or domains of anintegrin α heapiece polypeptide that interacts with an integrin βheadpiece polypeptide (as defined herein) to form a heterodimercomprising a ligand-binding site. The wild-type integrin α polypeptidesequences (comprising the ligand-binding headpiece and other domainsthat form the lower leg including, e.g., calf-1 and calf-2 domains) ofvarious species are available on the world wide web from the NCBI,including human and mouse. For example, the amino acid sequences ofhuman integrin α polypeptides and the corresponding nucleotide sequencesencoding the integrin α polypeptides are available at NCBI under GInumbers shown in Table 1 below. A skilled artisan can identify anintegrin α headpiece sequence from the corresponding integrin αpolypeptide/polynucleotide sequence, based on the known locations of,e.g., β-propeller domains, and ligand-binding sites, within thesequence.

TABLE 1 Sequences of human integrin α polypeptide subunits Humanintegrin α Amino acid sequence Nucleotide sequence polypeptide (GInumber) (GI number) α₁ 187957526 (SEQ ID NO: 7) 187957525 (SEQ ID NO:25) α₂  21105795 (SEQ ID NO: 8)  21105794 (SEQ ID NO: 26) α₃   186497(SEQ ID NO: 9)   186496 (SEQ ID NO: 27) α₄   903744 (SEQ ID NO: 10)  903743 (SEQ ID NO: 28) α₅  14250644 (SEQ ID NO: 11)  33870036 (SEQ IDNO: 29) α₆   33944 (SEQ ID NO: 12)   33943 (SEQ ID NO: 30) α₇  2897116(SEQ ID NO: 13)  2897115 (SEQ ID NO: 31) α₈  41393678 (SEQ ID NO: 14) 41393677 (SEQ ID NO: 32) α₉  52485941 (SEQ ID NO: 15)  52485940 (SEQ IDNO: 33) α₁₀  6650628 (SEQ ID NO: 16)  6650627 (SEQ ID NO: 34) α₁₁ 52485853 (SEQ ID NO: 17)  52485852 (SEQ ID NO: 35) α_(D)  62548866 (SEQID NO: 18)  62548865 (SEQ ID NO: 36) α_(E) 148728188 (SEQ ID NO: 19)148728187 (SEQ ID NO: 37) α_(L)  37515696 (SEQ ID NO: 20)  37515695 (SEQID NO: 38) α_(M)  68563402 (SEQ ID NO: 21) 109730155 (SEQ ID NO: 39)α_(V) 187953251 (SEQ ID NO: 22) 187953250 (SEQ ID NO: 40) α_(2B)116497075 (SEQ ID NO: 23) 116497074 (SEQ ID NO: 41) α_(X) 556503454 (SEQID NO: 24) 556503453 (SEQ ID NO: 42)

As used herein and throughout the specification, the term “functionalvariant” refers to a molecule, e.g., a polypeptide, that retains atleast about 70% or more (including at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) ofthe biological activity of a wild-type molecule, e.g., interaction withan integrin α headpiece polypeptide or an integrin β headpiecepolypeptide to form a heterodimer comprising a ligand-binding site thatretains (e.g., at least 70% or more of) the biological activity of thewild-type heterodimer, including, but not limited to, ligand binding,cellular adhesion, migration, invasion, differentiation, proliferation,apoptosis, and/or gene expression. The term “functional variant” as usedherein also encompasses conservative substitution variants of apolypeptide that retain at least about 70% or more (including at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99%, or 100%) of the biological activity of a wild-typemolecule, e.g., interaction with an integrin α headpiece polypeptide oran integrin β headpiece polypeptide to form a heterodimer comprising aligand-binding site that retains (e.g., at least 70% or more of) thebiological activity of the wild-type heterodimer, including, but notlimited to, ligand binding, cellular adhesion, migration, invasion,differentiation, proliferation, apoptosis, and/or gene expression.

“Conservative” amino acid substitutions can be made on the basis ofsimilarity in any of a variety or properties such as side chain size,polarity, charge, solubility, hydrophobicity, hydrophilicity, and/oramphipathicity of the residues involved. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,glycine, proline, phenylalanine, tryptophan and methionine. The polar(hydrophilic), neutral amino acids include serine, threonine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. In some embodimentscysteine is considered a non-polar amino acid. In some embodimentsinsertions or deletions may range in size from about 1 to 20 aminoacids, e.g., 1 to 10 amino acids. In some instances larger domains maybe removed without substantially affecting function.

The amino acid identity between two polypeptides can be determined, forexample, by first aligning the two polypeptide sequences using analignment algorithm, such as BLAST® or by other methods well-known inthe art.

In some embodiments, a modified integrin α headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α_(v) headpiece polypeptide or a functionalvariant thereof, with at least one Cys residue introduced into.

Accordingly, in one aspect, provided herein is a modified integrin α_(V)headpiece polypeptide. The modified integrin α_(V) headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α_(V) headpiece polypeptide (SEQ ID NO: 1) or afunctional variant thereof, with at least one Cys residue introducedthereto by one or more (e.g., at least one, at least two or more) of thefollowing modifications (a)-(e):

-   -   a. substitution of amino acid residues 399-401 (Ser-Met-Pro)        with one of the following: (i) Ser-Cys-Pro; (ii)        Gly-Cys-Pro; (iii) Ser-Cys-Gly; (iv) Gly-Cys-Gly; (v)        Ser-Gly-Cys-Pro (SEQ ID NO: 59); (vi) Ser-Cys-Gly-Pro (SEQ ID        NO: 60); (vii) Gly-Cys-Gly-Pro (SEQ ID NO: 61); and (viii)        Ser-Gly-Cys-Gly (SEQ ID NO: 62).    -   b. substitution of amino acid residues 310-311 (Gln-Glu) with        Gly-Cys;    -   c. substitution of amino acid residues 299 (Leu) and 310 (Gln)        with Cys and Gly, respectively;    -   d. substitution of amino acid residues 302-311        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln-Glu) (SEQ ID NO: 63) with        Gly-Gln-Gly-Cys (SEQ ID NO: 64); and    -   e. substitution of amino acid residue 299 (Leu) to Cys and        substitution of amino acid residues 302-310        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln) (SEQ ID NO: 65) with        Gly-Gln-Gly.

As used herein, the term “integrin α_(V) headpiece polypeptide” refersto a polypeptide that is similar or identical to the sequence of awild-type integrin α_(V) polypeptide having at least the headpiecethereof (including, e.g., β-propeller and/or thigh domains). In someembodiments, the term “integrin α_(V) headpiece polypeptide” refers to apolypeptide having an amino acid sequence that is at least 70% or more(including at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100%) identical to that of awild-type integrin α_(V) polypeptide having at least the headpiecethereof (including, e.g., β-propeller and/or thigh domains), and iscapable of interacting with an integrin β subunit to form a heterodimerand retaining (e.g., at least 70% or more of) the biological activity ofthe wild-type heterodimer, including, but not limited to,ligand-binding, cellular adhesion, migration, invasion, differentiation,proliferation, apoptosis, and/or gene expression. In some embodiments,the term “integrin α_(V) headpiece polypeptide” refers to a polypeptidethat is similar or identical to the sequence of a wild-type integrinα_(V) headpiece polypeptide (including, e.g., β-propeller and/or thighdomains). In some embodiments, the integrin α_(V) headpiece polypeptidecan further include other domain(s) of a wild-type integrin α_(V)polypeptide such as calf-1 and/or calf-2 domains that form the lowerlegs of the wild-type integrin α_(V) polypeptide. In some embodiments,an integrin α_(V) headpiece polypeptide refers to a full-lengthwild-type integrin α_(V) headpiece polypeptide sequence. In someembodiments, an integrin α_(V) headpiece polypeptide refers to afunctional domain or domains (e.g., β-propeller and/or thigh domains) ofan integrin α_(V) headpiece polypeptide that interacts with an integrinβ headpiece polypeptide (as defined herein) to form a heterodimercomprising a ligand-binding site. The wild-type integrin α_(V) headpiecesequences of various species can be identified from the full-lengthintegrin α_(V) sequences available on the world wide web from the NCBI,including human (See Table 1 above) and mouse. In one embodiment, theintegrin α_(V) headpiece polypeptide comprises, consists essentially of,or consists of the amino acid sequence of human integrin α_(V) subunitshown in SEQ ID NO: 1.

In some embodiments, the modified integrin α_(V) headpiece polypeptidecan comprise, consist essentially of, or consist of an amino acidsequence of a human integrin α_(V) headpiece polypeptide (e.g., of SEQID NO: 1) or a functional variant thereof, with at least one Cys residueintroduced thereto by substitution of amino acid residues 399-401(Ser-Met-Pro) with Ser-Cys-Pro (modification (a)(i)). In someembodiments, the modified integrin α_(V) headpiece polypeptide cancomprise, consist essentially of, or consist of an amino acid sequenceof an integrin α_(V) headpiece polypeptide (e.g., of SEQ ID NO:1) or afunctional variant thereof, with at least one Cys residue introducedthereto by substitution of amino acid residues 399-401 (Ser-Met-Pro)with Gly-Cys-Pro (modification (a)(ii)). In some embodiments, themodified integrin α_(V) headpiece polypeptide can comprise, consistessentially of, or consist of an amino acid sequence of an integrinα_(V) headpiece polypeptide (e.g., of SEQ ID NO:1) or a functionalvariant thereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withSer-Gly-Cys-Pro (SEQ ID NO: 59) (modification (a)(v)).

In some embodiments, the modified integrin α_(V) headpiece polypeptidecan comprise, consist essentially of, or consist of an amino acidsequence of an integrin α_(V) headpiece polypeptide (e.g., of SEQ IDNO: 1) or a functional variant thereof, with at least two Cys residuesintroduced thereto by at least two or more of the modifications (a)-(e)as described above. By way of example only, in some embodiments, atleast two Cys residues can be introduced into the modified integrinα_(V) headpiece polypeptide by (1) one of the modifications (a)(i)-(viii) as described above; and (2) at least one of the modifications(b)-(e) as described above.

In some embodiments, the modified integrin α headpiece polypeptides(e.g., modified integrin α_(V) polypeptides) are soluble polypeptides.The term “soluble” as used herein and throughout the specificationgenerally refers to dissolution of a molecule, e.g., a polypeptide, in afluid (e.g., a liquid, a solution, and/or a mixture) under a specifiedcondition, e.g., characterized by a number of factors, including, e.g.,temperature, pressure, ion concentration, and/or pH. The dissolution canbe partial or complete. As used in reference to the modified integrin αand/or β headpiece polypeptides described herein, the term “soluble”refers to complete or partial dissolution of the integrin headpiecepolypeptides in an aqueous buffered solution at a specified temperature(e.g., at room temperature). The aqueous buffered solution can furthercomprise metal ions (e.g., Ca²⁺, Na⁺ and/or Mg²⁺). Alternatively oradditionally, the term “soluble” in reference to modified integrin αand/or β headpiece polypeptides described herein means that thepolypeptides are not expressed on cell surface.

In some embodiments, the modified integrin α headpiece polypeptides(e.g., modified integrin α_(v) headpiece polypeptides) can be isolatedor purified. As used herein and throughout the specification, the term“isolated” with respect to a polypeptide or protein means a polypeptideor protein in a form that is relatively free from material such ascontaminating molecules, including, e.g., polypeptides, lipids, nucleicacids and other cellular material that is normally is associated withthe polypeptide or protein in a cell or that is associated with thepolypeptide or protein in a library or in a crude preparation.

The term “purified” as applied to polypeptides or proteins herein refersto a composition comprising a desired polypeptide or protein in at least50% or more of the total protein components in the composition. In someembodiments, the composition comprises a desired polypeptide or proteinin at least 60% or more (including, e.g., at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 99%, or 100%) of the total protein components in thecomposition. The composition can comprise other non-protein componentssuch as carbohydrates, and/or salts. In some embodiments, thecomposition can comprise, consist essentially of, or consist of adesirable polypeptide or protein in a buffered solution. For example,the modified integrin α headpiece polypeptides (e.g., modified integrinα_(v) headpiece polypeptides) can be present in a buffered solution.

In some embodiments, the modified integrin α headpiece polypeptides(e.g., modified integrin α_(V) headpiece polypeptides) can furthercomprise a detectable label described herein.

In some embodiments, the modified integrin α headpiece polypeptides(e.g., modified integrin α_(V) headpiece polypeptides) can be furtherattached to a solid surface. Depending on the need of desiredapplications, the solid surface can be made of any material, including,but are not limited to, glass, silicone, cellulose-based materials(e.g., paper), plastics, polymer, and/or any combinations thereof.

Polynucleotides encoding the modified integrin α headpiece polypeptides(e.g., the modified integrin α_(V) headpiece polypeptides describedherein) are also encompassed within the scope of the inventionsdescribed herein.

Modified Integrin β Headpiece Polypeptides (e.g., Modified Integrin β₆headpiece polypeptides)

Another aspect provided herein relates to a modified integrin βheadpiece polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence of an integrin β headpiecepolypeptide or a functional variant thereof, with at least one or more(e.g., at least one, at least two, at least three or more) Cys residuesintroduced into.

In some embodiments, Cys residue(s) can be introduced into domain(s) ofan integrin β headpiece polypeptide that are distal from aligand-binding site. The modified integrin β headpiece polypeptide cansubstantially retain the functionality (e.g., ligand-binding capability)of the naturally occurring integrin β headpiece polypeptide (e.g.,retain at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 99% or more and up to 100%), while having theability to form a disulfide bond with an integrin α polypeptide or amodified integrin α headpiece polypeptide.

A Cys residue can be introduced into one or more domain(s) of anintegrin β headpiece polypeptide by any methods of modifying amino acidsknown in the art, including, e.g., substitution, deletion and/oraddition of an amino acid or amino acid analog. In some embodiments, atleast one or more Cys residue or an analog thereof can be introducedinto one or more domain(s) of the integrin β headpiece polypeptide bysubstitution of an amino acid residue, and/or addition of a Cys residueor an analog thereof. In some embodiments, while introducing one or moreCys residue into at least one domain of the integrin β headpiecepolypeptide, one or more other amino acid residues present in theintegrin β headpiece polypeptide can also be deleted.

As used herein, the term “integrin β headpiece polypeptide” refers to apolypeptide that is similar or identical to the sequence of wild-typeintegrin β polypeptide having at least the headpiece thereof Δn integrinβ headpiece polypeptide generally includes a βI domain, a hybrid domain,a PSI (plexin, semaphoring, and integrin) domain, and/or an I-EGF-1domain, and confers ligand-binding capability upon binding with anappropriate integrin α headpiece polypeptide. An integrin β headpiecepolypeptide can be derived from the headpiece of any integrin βpolypeptide, including, for example, β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈.In some embodiments, the term “integrin β polypeptide” refers to apolypeptide having an amino acid sequence that is at least 70% or more(including at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100%) identical to that of awild-type integrin β polypeptide having at least the headpiece thereof(including, e.g., βI, hybrid, PSI and/or I-EGF-1 domains), and iscapable of interacting with an integrin α subunit (e.g., a headpiece) toform a heterodimer and retaining (e.g., at least 70% or more of) thebiological activity of the wild-type heterodimer, including, but notlimited to, ligand binding, cellular adhesion, migration, invasion,differentiation, proliferation, apoptosis, and/or gene expression. Insome embodiments, the term “integrin β headpiece polypeptide” refers toa polypeptide that is similar or identical to the sequence of awild-type integrin β headpiece polypeptide (including, e.g., βI, hybrid,PSI and/or I-EGF-1 domains). In some embodiments, the integrin βheadpiece polypeptide can further include other domain(s) of a wild-typeintegrin β polypeptide such as I-EGF-2 to I-EGF-4 and/or β tail domainsthat form the lower legs of the wild-type integrin β polypeptide. Insome embodiments, an integrin β headpiece polypeptide refers to afull-length wild-type integrin β headpiece polypeptide sequence. In someembodiments, an integrin β headpiece polypeptide refers to a functionaldomain or domains of an integrin β headpiece polypeptide that interactswith an integrin α headpiece polypeptide to form a heterodimercomprising a ligand-binding site. The wild-type integrin β polypeptidesequences (comprising the ligand-binding headpiece and other domainsthat form the lower leg, including, e.g., I-EGF-2 to I-EGF-4 and/or βtail domains) of various species are available on the world wide webfrom the NCBI, including human and mouse. For example, the amino acidsequences of human integrin β polypeptides and the correspondingnucleotide sequences encoding the integrin β polypeptides are availableat NCBI under GI numbers shown in Table 2 below. A skilled artisan canidentify an integrin β headpiece sequence from the correspondingintegrin β polypeptide/polynucleotide sequence, based on the knownlocations of, e.g., β1 domain, and/or ligand-binding sites, within thesequence.

TABLE 2 Sequences of human integrin β polypeptides Human integrin βAmino acid sequence Nucleotide sequence polypeptide (GI number) (GInumber) β₁ 218563324 (SEQ ID NO: 43) 182519230 (SEQ ID NO: 51) β₂  825636 (SEQ ID NO: 44)   186508 (SEQ ID NO: 52) β₃ 119578086 (SEQ IDNO: 45) 118341516 (SEQ ID NO: 53) β₄   33951 (SEQ ID NO: 46)   33950(SEQ ID NO: 54) β₅   306894 (SEQ ID NO: 47)   184524 (SEQ ID NO: 55) β₆119631795 (SEQ ID NO: 48) 115527926 (SEQ ID NO: 56) β₇   186511 (SEQ IDNO: 49)   186510 (SEQ ID NO: 57) β₈   184521 (SEQ ID NO: 50)   184520(SEQ ID NO: 58)

In some embodiments, a modified integrin β headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin β₆ headpiece polypeptide or a functional variantthereof, with at least one Cys residue introduced into.

Accordingly, in one aspect, provided herein is a modified integrin β₆headpiece polypeptide. The modified integrin β₆ headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin β₆ headpiece polypeptide (SEQ ID NO: 2) or afunctional variant thereof, with at least one or more (e.g., at leastone, at least two, at least three or more) Cys residues introducedthereto by one, two or all three of the following modifications (f)-(h):

-   -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.

As used herein, the term “integrin β₆ headpiece polypeptide” refers to apolypeptide that is similar or identical to the sequence of wild-typeintegrin β₆ headpiece polypeptide having at least the headpiece thereof(including, e.g., βI, hybrid, PSI, and/or I-EGF-1 domains). In someembodiments, the term “integrin β₆ headpiece polypeptide” refers to apolypeptide having an amino acid sequence that is at least 70% or more(including at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100%) identical to that of awild-type integrin β₆ headpiece polypeptide (including, e.g., βI,hybrid, PSI, and/or I-EGF-1 domains), and is capable of interacting withan integrin α headpiece polypeptide to form a heterodimer and retaining(e.g., at least 70% or more of) the biological activity of the wild-typeheterodimer, including, but not limited to, ligand binding, cellularadhesion, migration, invasion, differentiation, proliferation,apoptosis, and/or gene expression. In some embodiments, the term“integrin β₆ headpiece polypeptide” refers to a polypeptide that issimilar or identical to the sequence of a wild-type integrin β₆headpiece polypeptide (including, e.g., βI, hybrid, PSI, and/or I-EGF-1domains). In some embodiments, the integrin β₆ headpiece polypeptide canfurther include other domain(s) of a wild-type integrin α_(v)polypeptide such as I-EGF-2, I-EGF-3, I-EGF-4, and/or β tail domainsthat form the lower legs of the wild-type integrin β₆ polypeptide. Insome embodiments, an integrin β₆ polypeptide refers to a full-lengthwild-type integrin β₆ polypeptide sequence. In some embodiments, anintegrin β₆ headpiece polypeptide refers to a functional domain ordomains of an integrin β₆ headpiece polypeptide that interacts with anintegrin α headpiece polypeptide to form a heterodimer comprising aligand-binding site. The wild-type integrin β₆ headpiece sequences ofvarious species can be identified from the full-length integrin β₆sequences available on the world wide web from the NCBI, including human(see Table 2 above) and mouse. In one embodiment, the integrin β₆headpiece polypeptide comprises, consists essentially of, or consists ofthe amino acid sequence of human integrin β₆ headpiece polypeptide shownin SEQ ID NO:2.

In some aspects, provided herein are fragments of the modified integrinβ₆ headpiece polypeptides described herein. In one aspect, the integrinβ₆ headpiece polypeptide fragment comprises, consists essentially of, orconsists of a βI domain of integrin β₆ subunit with at least one Cysresidue introduced thereto by one, two, or all of the followingmodifications (f)-(h), the βI domain is defined from residues DYP toresidues ELR as shown in an amino acid sequence of SEQ ID NO: 2, or afunctional variant thereof.

(f) substitution of amino acid residue 270 (Ile) with Cys;

(g) substitution of amino acid residue 294 (Thr) with Cys; and

(h) substitution of amino acid residue 296 (Gly) with Cys.

In another aspect, the integrin β₆ headpiece polypeptide fragmentcomprises, consists essentially of, or consists of a βI domain and ahybrid domain of integrin β₆ subunit, the βI domain being defined fromresidues DYP to residues ELR as shown in an amino acid sequence of SEQID NO: 2, or a functional variant thereof, while the hybrid domain beingdefined from residues ENP to residues QTE, and/or from residues SEV toresidues ECN as shown in an amino acid sequence of SEQ ID NO: 2, or afunctional variant thereof; wherein at least one least one Cys residueis introduced to the βI domain by one, two, or all of the followingmodifications (f)-(h):

(f) substitution of amino acid residue 270 (Ile) with Cys;

(g) substitution of amino acid residue 294 (Thr) with Cys; and

(h) substitution of amino acid residue 296 (Gly) with Cys.

In yet another aspect, the integrin β₆ headpiece polypeptide fragmentcomprises, consists essentially of, or consists of a βI domain, a hybriddomain, and a PSI domain of integrin β₆ subunit, the βI domain beingdefined from residues DYP to residues ELR as shown in an amino acidsequence of SEQ ID NO: 2, or a functional variant thereof, while thehybrid domain being defined from residues ENP to residues QTE, and/orfrom residues SEV to residues ECN as shown in an amino acid sequence ofSEQ ID NO: 2, or a functional variant thereof; and the PSI domain beingdefined from from residues HVQ to residues NFI as shown in an amino acidsequence of SEQ ID NO: 2; wherein at least one least one Cys residue isintroduced to the βI domain by one, two, or all of the followingmodifications (f)-(h):

(f) substitution of amino acid residue 270 (Ile) with Cys;

(g) substitution of amino acid residue 294 (Thr) with Cys; and

(h) substitution of amino acid residue 296 (Gly) with Cys.

The modified integrin β₆ headpiece polypeptide fragments of variousaspects described herein can be isolated. The modified integrin β₆headpiece polypeptide fragments of various aspects described herein canfurther be attached to a solid surface.

In some embodiments, the modified integrin β₆ headpiece polypeptidefragments of various aspects described herein are soluble polypeptides.

In another aspect, provided herein is a modified integrin β₃ headpiecepolypeptide. The integrin β₃ headpiece polypeptide comprises,essentially consist of, or consist of amino acid residues 27 to 498 ofSEQ ID NO: 5 or a functional fragment thereof (e.g., with desireddomain(s)) with at least one Cys residue introduced thereto bysubstitution of amino acid residue 293 (Gln) with Cys. The modifiedintegrin β₃ headpiece polypeptide can be isolated. The modified integrinβ₃ headpiece polypeptide can further be attached to a solid surface.

In another aspect, provided herein is a modified integrin β₈ headpiecepolypeptide. The integrin β₈ headpiece polypeptide comprises,essentially consist of, or consist of amino acid residues 43 to 498 ofSEQ ID NO: 6 or a functional fragment thereof (e.g., with desireddomain(s)) with at least one Cys residue introduced thereto bysubstitution of amino acid residue 301 (Val) with Cys. The modifiedintegrin β₈ headpiece polypeptide can be isolated. The modified integrinβ₈ headpiece polypeptide can further be attached to a solid surface.

In some embodiments, the modified integrin β₆ headpiece polypeptide cancomprise, consist essentially of, or consist of an amino acid sequenceof an integrin β₆ headpiece polypeptide (SEQ ID NO: 2) or a functionalvariant thereof (e.g., with desired domain(s)), with at least one Cysresidue introduced thereto by substitution of amino acid residue 270(Ile) with Cys (modification (f)).

In some embodiments, the modified integrin β₆ headpiece polypeptide cancomprise, consist essentially of, or consist of an amino acid sequenceof an integrin β₆ headpiece polypeptide (SEQ ID NO: 2) or a functionalvariant thereof, with at least two Cys residues introduced thereto by atleast two or more of the modifications (f)-(h) as described above. Byway of example only, in some embodiments, at least two Cys residues canbe introduced into the modified integrin β₆ headpiece polypeptide by anytwo of the modifications (f)-(h) as described above.

In some embodiments, the modified integrin β headpiece polypeptides(e.g., modified integrin β₆ polypeptides) are soluble polypeptides.

In various embodiments, the modified integrin β headpiece polypeptides(e.g., β₆ headpiece polypeptides) can be isolated or purified. In someembodiments, the modified integrin β headpiece polypeptides (e.g., β₆headpiece polypeptides) can be present in a buffered solution.

In some embodiments, the modified integrin β headpiece polypeptides(e.g., β₆ headpiece polypeptides) can further comprise a detectablelabel described herein.

In various embodiments, the modified integrin β headpiece polypeptides(e.g., β₆ headpiece polypeptides) or functional variants/fragmentsthereof can be further attached to a solid surface. Depending on theneed of desired applications, the solid surface can be made of anymaterial, including, but are not limited to, glass, silicone,cellulose-based materials (e.g., paper), plastics, polymer, and/or anycombinations thereof.

Polynucleotides encoding the modified integrin β headpiece polypeptides(e.g., the modified integrin β₆ headpiece polypeptides described herein)or functional variants/fragments thereof are also encompassed within thescope of the inventions described herein.

Modified Integrin Polypeptide Dimers or Functional Fragments VariantsThereof

Modified integrin polypeptide dimers comprising at least one of themodified integrin α headpiece polypeptide and the modified integrin βheadpiece polypeptide are also provided herein. In accordance with someaspects described herein, the modified integrin polypeptide dimerscomprise at least one or more (e.g., at least one, at least two, atleast three or more) disulfide bonds linking the two integrin α and βheadpiece subunits or functional variants/fragments thereof. Themodified integrin polypeptide dimers described herein can still interactwith a natural ligand as it binds to a naturally occurring integrinheterodimer, but, unlike naturally occurring integrin heterodimers thatcan reversibly dissociate, these modified polypeptide dimers describedherein do not dissociate and enable formation of a crystallizablestructure of an integrin heterodimer, alone or complexed with a testligand, via formation of a disulfide bridge between the two integrinsubunits.

One aspect of the modified integrin polypeptide dimers provided hereinrelates to a modified integrin polypeptide dimer comprising, consistingessentially of, or consisting of a modified integrin α headpiecepolypeptide described herein, and an integrin β headpiece polypeptide,wherein the modified integrin α polypeptide and the integrin β headpiecepolypeptide are covalently linked by at least one (e.g., at least one,at least two, at least three or more) disulfide bonds. Generally, theintegrin α polypeptide can comprise, essentially of, or consist of aβ-propeller domain and a thigh domain. In some embodiments, the integrinβ polypeptide can comprise, essentially consist of, or consist of a βIdomain. In some embodiments, the integrin β polypeptide can comprise,essentially consist of, or consist of a βI domain and a hybrid domain.In some embodiments, the integrin β polypeptide can comprise,essentially consist of, or consist of a βI domain, a hybrid domain, anda PSI domain.

As described above, the modified integrin α headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α headpiece polypeptide (including, e.g.,β-propeller and/or thigh domains), with at least one or more (e.g., atleast one, at least two or more) Cys residues introduced thereto. Theintegrin α headpiece polypeptide can be derived from the headpiece ofthe corresponding integrin α polypeptide selected from the groupconsisting of α₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D),α_(E), α_(L), α_(M), α_(V), α₂B, and α_(X)) or a functional variantthereof.

In some embodiments, the modified integrin α headpiece polypeptide is amodified integrin α_(V) headpiece polypeptide described herein. In someembodiments, the modified integrin α_(V) headpiece polypeptide cancomprise, consist essentially of, or consist of a substitution of aminoacid residues 399-401 (Ser-Met-Pro) with Ser-Cys-Pro (modification (a)(i)). In some embodiments, the modified integrin α_(v) headpiecepolypeptide can comprise, consist essentially of, or consist of asubstitution of amino acid residues 399-401 (Ser-Met-Pro) withGly-Cys-Pro (modification (a) (ii)). In some embodiments, the modifiedintegrin α_(v) headpiece polypeptide can comprise, consist essentiallyof, or consist of a substitution of amino acid residues 399-401(Ser-Met-Pro) with Ser-Gly-Cys-Pro (SEQ ID NO: 59) (modification (a)(v)).

In various embodiments, the integrin β headpiece polypeptide covalentlylinked to a modified integrin α headpiece polypeptide described herein(e.g., a modified integrin α_(V) headpiece polypeptide described herein)can be derived from the full-length integrin β polypeptide selected fromthe group consisting of β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈.

Another aspect of the modified integrin polypeptide dimers providedherein relates to a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of an integrin α headpiecepolypeptide, and a modified integrin β headpiece polypeptide describedherein or a modified integrin β headpiece polypeptide fragment (e.g.,with desired domain(s)) described herein, wherein the integrin αheadpiece polypeptide and the modified integrin β headpiece polypeptideare covalently linked by at least one (e.g., at least one, at least two,at least three or more) disulfide bonds.

As described above, the modified integrin β headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin β headpiece polypeptide, with at least one ormore (e.g., at least one, at least two or more) Cys residues introducedthereto. The integrin β headpiece polypeptide can be derived from theheadpiece of the corresponding integrin β polypeptide selected from thegroup consisting of β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈) or a functionalvariant thereof.

In some embodiments, the modified integrin β polypeptide is a modifiedintegrin β₆ headpiece polypeptide described herein or a functionalvariant thereof (e.g., a βI domain alone, or in combination with ahybrid domain and/or a PSI domain). In some embodiments, the modifiedintegrin β₆ headpiece polypeptide or a functional variant thereof cancomprise, consist essentially of, or consist of a substitution of aminoacid residue 270 (Ile) with Cys (modification (f)). It should be notedthat numbering is based on SEQ ID NO: 2, which is the amino acidsequence of the β₆ full headpiece. One of skill in the art can adjustthe numbering of the corresponding cysteine substitution, e.g., whenonly a βI domain is used.

In some embodiments, the modified integrin β headpiece polypeptide is amodified integrin β₃ headpiece polypeptide described herein or afunctional variant thereof (e.g., a βI domain alone, or in combinationwith a hybrid domain and/or a PSI domain). In some embodiments, themodified integrin β₃ headpiece polypeptide or a functional variantthereof can comprise, consist essentially of, or consist of asubstitution of amino acid residue 293 (Gln) with Cys. It should benoted that numbering is based on SEQ ID NO: 5, which is the amino acidsequence of the β₃ full headpiece. One of skill in the art can adjustthe numbering of the corresponding cysteine substitution, e.g., whenonly a βI domain is used.

In some embodiments, the modified integrin β headpiece polypeptide is amodified integrin β₈ headpiece polypeptide described herein or afunctional variant thereof (e.g., a βI domain alone, or in combinationwith a hybrid domain and/or a PSI domain). In some embodiments, themodified integrin β₈ headpiece polypeptide or a functional variantthereof can comprise, consist essentially of, or consist of asubstitution of amino acid residue 301 (Val) with Cys. It should benoted that numbering is based on SEQ ID NO: 6, which is the amino acidsequence of the β₈ full headpiece. One of skill in the art can adjustthe numbering of the corresponding cysteine substitution, e.g., whenonly a βI domain is used.

In some embodiments, the integrin α headpiece polypeptide covalentlylinked to the modified integrin β headpiece polypeptide can be derivedfrom the headpiece of the corresponding integrin α polypeptide selectedfrom the group consisting of α₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀,α₁₁, α_(D), α_(E), α_(L), α_(M), α_(V), α_(2B), and α_(X). In someembodiments where the modified integrin β polypeptide is a modifiedintegrin β₆ headpiece polypeptide described herein, the integrin αheadpiece polypeptide covalently linked to the modified integrin β₆headpiece polypeptide is derived from the headpiece of the integrinα_(V) polypeptide.

A further aspect of the modified integrin polypeptide dimers providedherein relates to a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of a modified integrin αheadpiece polypeptide described herein or a functional variant thereof,and a modified integrin β headpiece polypeptide described herein or afunctional variant thereof, wherein the modified integrin α headpiecepolypeptide or a functional variant thereof and the modified integrin βheadpiece polypeptide or a functional variant thereof are covalentlylinked by at least one (e.g., at least one, at least two, at least threeor more) disulfide bonds.

The modified integrin α headpiece polypeptide or a functional variantthereof comprises, consists essentially of, or consists of an amino acidsequence of an integrin α headpiece polypeptide (for example, theheadpiece of one of the integrin α polypeptide selected from the groupconsisting of α₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D),α_(E), α_(L), α_(M), α_(V), α_(2B), and α_(X)) or a functional variantthereof, with at least one or more (e.g., at least one, at least two ormore) Cys residues introduced thereto; while the modified integrin βheadpiece polypeptide comprises, consists essentially of, or consists ofan amino acid sequence of an integrin β headpiece polypeptide (forexample, the headpiece of one of the integrin β polypeptide selectedfrom the group consisting of β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈) or afunctional variant thereof, with at least one or more (e.g., at leastone, at least two or more) Cys residues introduced thereto.

In one aspect, provided herein is a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α₅ headpiece polypeptide and a modified integrin β₁ headpiecepolypeptide covalently linked together by at least one or more disulfidebond. In one embodiment, the modified integrin α₅ headpiece polypeptidecomprises, consists essentially of, or consists of an amino acidsequence of an integrin α₅ headpiece polypeptide (e.g., SEQ ID NO: 3)with at least one Cys residue introduced thereby by substitution ofamino acid residue 452 (Thr) with Cys. (SEQ ID NO: 3 includes the signalpeptide sequence at positions 1-41. Without the signal peptide sequence,the numbering of the amino acid residue Thr being substituted with Cyswould become amino acid residue 411). In one embodiment, the modifiedintegrin β₁ headpiece polypeptide comprises, consists essentially of, orconsists of one, two, three, or all domains of an integrin pi headpiecepolypeptide (e.g., SEQ ID NO: 4) selected from the group consisting of aPSI domain, hybrid domain, βI domain, and an EGF-1 domain, with at leastone Cys residue introduced thereby by substitution of amino acid residue295 (Leu) with Cys. (SEQ ID NO: 4 includes the signal peptide sequenceat positions 1-20. Without the signal peptide sequence, the numbering ofthe amino acid residue Leu being substituted with Cys would become aminoacid residue 275).

In one aspect, a modified integrin polypeptide dimer comprising,consisting essentially of, or consisting of a modified integrin α_(v)headpiece polypeptide described herein and a modified integrin β₃headpiece polypeptide covalently linked together by at least onedisulfide bond is also provided herein. In one embodiment, the modifiedintegrin β₃ headpiece polypeptide comprises, consists essentially of, orconsists of one, two, three, or all domains of an integrin β₃ headpiecepolypeptide (e.g., SEQ ID NO: 5) selected from the group consisting of aPSI domain, hybrid domain, βI domain, and an EGF-1 domain, with at leastone Cys residue introduced thereby by substitution of amino acid residue293 (Gln) with Cys. (SEQ ID NO: 5 includes the signal peptide sequenceat positions 1-26. Without the signal peptide sequence, the numbering ofthe amino acid residue Gln being substituted with Cys would become aminoacid residue 267).

One aspect provided herein relates to a modified integrin polypeptidedimer comprising, consisting essentially of, or consisting of a modifiedintegrin α_(v) headpiece polypeptide described herein and a modifiedintegrin β₈ headpiece polypeptide covalently linked together by at leastone disulfide bond. In one embodiment, the modified integrin β₈headpiece polypeptide comprises, consists essentially of, or consists ofone, two, three, or all domains of an integrin β₈ headpiece polypeptide(e.g., SEQ ID NO: 6) selected from the group consisting of a PSI domain,hybrid domain, βI domain, and an EGF-1 domain, with at least one Cysresidue introduced thereby by substitution of amino acid residue 301(Val) with Cys. (SEQ ID NO: 6 includes the signal peptide sequence atpositions 1-42. Without the signal peptide sequence, the numbering ofthe amino acid residue Val being substituted with Cys would become aminoacid residue 259).

A further aspect provides a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α_(v) headpiece polypeptide described herein and a modifiedintegrin β₆ headpiece polypeptide covalently linked together by at leastone disulfide bond. The modified integrin polypeptide dimer comprises,consists essentially of, or consists of (i) an integrin α_(V) headpiecepolypeptide or a functional variant thereof, with at least one or more(e.g., at least one, at least two or more) Cys residues introducedthereto; and (ii) an integrin β₆ headpiece polypeptide or a functionalvariant thereof, with at least one or more (e.g., at least one, at leasttwo or more) Cys residues introduced thereto.

Table 3 below lists some embodiments of the modified integrinpolypeptide dimers described herein comprising, consisting essentiallyof, or consisting of a modified integrin α_(v) headpiece polypeptidedescribed herein and a modified integrin β₆ headpiece polypeptidedescribed herein. By way of example only, as shown in Table 3, any oneof the modified integrin α_(v) headpiece polypeptide (comprising,consisting essentially of, or consisting of at least one modification(a) (i) to (c)) can form a modified integrin polypeptide dimer with oneof the modified integrin β₆ headpiece polypeptides (selected frommodifications (f) to (h)) as indicated by a “x” symbol in the table. Insome embodiments, a modified integrin α_(v) headpiece polypeptidecomprising at least two modifications selected from (a) (i) to (c)provided that there is no overlap in the modifications can form amodified integrin polypeptide dimer with at least one or more (e.g., atleast one, at least two or more) of the modified integrin β6 headpiecepolypeptides (selected from modifications (f) to (h)).

Table 3: Exemplary combinations of the modified integrin polypeptidedimers described herein comprising, consisting essentially of, orconsisting of a modified integrin α_(v) headpiece polypeptide describedherein and a modified integrin β₆ headpiece polypeptide describedherein. Table discloses SEQ ID NOS 60-65, respectively, in order ofappearance.

Based on SEQ ID NO: 2 f g h Substitution Substitution Substitution ofamino of amino of amino acid residue acid residue acid residue 270 (Ile)294 (Thr) 296 (Gly) Modifications with Cys with Cys with Cys SEQ ID NO:1 a(i) Substitution of amino acid x x x residues 399-401 (Ser-Met-Pro)with Ser-Cys-Pro a(ii) Substitution of amino acid x x x residues 399-401(Ser-Met-Pro) with Gly-Cys-Pro a(iii) Substitution of amino acid x x xresidues 399-401 (Ser-Met-Pro) with Ser-Cys-Gly a(iv) Substitution ofamino acid x x x residues 399-401 (Ser-Met-Pro) with Gly-Cys-Gly a(v)Substitution of amino acid x x x residues 399-401 (Ser-Met-Pro) withSer-Gly-Cys-Pro a(vi) Substitution of amino acid x x x residues 399-401(Ser-Met-Pro) with Ser-Cys-Gly-Pro a(vii) Substitution of amino acid x xx residues 399-401 (Ser-Met-Pro) with Gly-Cys-Gly-Pro a(viii)Substitution of amino acid x x x residues 399-401 (Ser-Met-Pro) withSer-Gly-Cys-Gly b Substitution of amino acid x x x residues 310-311(Gln-Glu) with Gly-Cys c Substitution of amino acid x x x residues 299(Lys) and 310 (Gln) with Cys and Gly, respectively d Substitution ofamino acid x x x residues 302-311 (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln-Glu) with Gly-Gln-Gly-Cys e Substitution ofamino acid x x x residue 299 (Lys) with Cys and Substitution of aminoacid residues 302-310 (Asp-Arg-Gly- Ser-Asp-Gly-Lys-Leu-Gln) withGly-Gln-Gly

In one aspect, provided herein is a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α_(v) headpiece polypeptide and a modified integrin β₆headpiece polypeptide covalently linked together by at least one or moredisulfide bonds, wherein:

the modified integrin α_(v) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(V) polypeptide (SEQ ID NO: 1) or a functional variant thereof, withat least one Cys residue introduced thereto by substitution of aminoacid residues 399-401 (Ser-Met-Pro) with Ser-Cys-Pro (modification(a)(i)); and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆polypeptide (SEQ ID NO: 2) or a functional variant thereof, with atleast one Cys residue introduced thereto by substitution of amino acidresidue 270 (Ile) with Cys (modification (f)).

In one aspect, provided herein is a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α_(V) headpiece polypeptide and a modified integrin β₆headpiece polypeptide covalently linked together by at least one or moredisulfide bonds, wherein:

the modified integrin α_(v) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(v) headpiece polypeptide (SEQ ID NO: 1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withGly-Cys-Pro (modification (a)(ii)); and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by substitution ofamino acid residue 270 (Ile) with Cys (modification (f)).

In one aspect, provided herein is a modified integrin polypeptide dimercomprising, consisting essentially of, or consisting of a modifiedintegrin α_(v) headpiece polypeptide and a modified integrin β₆headpiece polypeptide covalently linked together by at least one or moredisulfide bonds, wherein:

the modified integrin α_(v) headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrinα_(v) headpiece polypeptide (SEQ ID NO: 1) or a functional variantthereof, with at least one Cys residue introduced thereto bysubstitution of amino acid residues 399-401 (Ser-Met-Pro) withSer-Gly-Cys-Pro (SEQ ID NO: 59) (modification (a)(v)); and

the modified integrin β₆ headpiece polypeptide comprises, consistsessentially of, or consists of an amino acid sequence of an integrin β₆headpiece polypeptide (SEQ ID NO: 2) or a functional variant thereof,with at least one Cys residue introduced thereto by substitution ofamino acid residue 270 (Ile) with Cys (modification (f)).

In some embodiments of various aspects described herein, the modifiedintegrin polypeptide dimer can be soluble polypeptide dimer.

In some embodiments of various aspects described herein, the modifiedintegrin polypeptide dimer can be isolated or purified. In someembodiments, the modified integrin polypeptide dimer (e.g., modifiedintegrin α_(V)β₆ polypeptide dimer) can be present in a bufferedsolution.

In some embodiments of various aspects described herein, the modifiedintegrin polypeptide dimer can further comprise a detectable labeldescribed herein.

The modified integrin polypeptide dimer can further be attached to asolid surface. Depending on the need of desired applications, the solidsurface can be made of any material, including, but are not limited to,glass, silicone, cellulose-based materials (e.g., paper), plastics,polymer, and/or any combinations thereof.

Exemplary Methods of Using the Modified Integrin α and or 8 HeadpiecePolypeptides or Modified Integrin Polypeptide Dimers Described Herein

The modified integrin α headpiece polypeptides described herein, themodified integrin β headpiece polypeptides described herein, and themodified integrin polypeptide dimers described herein can still interactwith a natural ligand as it binds to a naturally occurring integrinheterodimer, but, unlike naturally occurring integrin heterodimers thatcan reversibly dissociate, the modified integrin polypeptide dimersdescribed herein do not dissociate and enable formation of acrystallizable structure of an integrin heterodimer, alone or complexedwith a test ligand, via formation of a disulfide bridge between the twointegrin subunits. Accordingly, in some aspects, the compositions asdescribed herein can be used to characterize an integrin-ligandinteraction, e.g., to measure the binding affinity of a test agent to anintegrin heterodimer (e.g., integrin α_(v)β₆ heterodimer); and/or toidentify a novel integrin ligand, e.g., in a drug discovery process.

For example, a method for determining whether a test agent forms acomplex with an integrin heterodimer is provided herein. The methodcomprises contacting one or more of the modified integrin polypeptidedimers described herein with a test agent, and detecting formation of acomplex comprising the modified integrin polypeptide dimer and the testagent bound thereto. Detection of a formed complex comprising themodified integrin polypeptide dimer and the test agent bound theretoindicates that the test agent is capable of forming a complex with theintegrin.

Various methods known in the art can be used to detect formation of acomplex comprising the modified integrin polypeptide dimer and a testagent bound thereto. By way of example only, in some embodiments, thecomplex can be detected by a detection method comprising crystallizationof the complex. As used herein, the term “crystallization” refers to theprocess of formation of a crystal. In some embodiments, the term“crystallization” refers to the process of formation of a proteincrystal. The quality of protein crystallization can vary with a numberof factors including, e.g., sensitivity of protein samples totemperature, pH, ionic strength, and other factors. Determination ofappropriate crystallization conditions for a given protein oftenrequires empirical testing of various conditions. Various methods ofprotein crystallization are known in the art and can be used in themethods described to cystallize the complex. Exemplary methods ofprotein crystallization include, but are not limited to vapor diffusion,microbatch, microdialysis (via a semi-permeable membrane, across whichsmall molecules and ions can pass, while proteins and large polymerscannot pass), free-interface diffusion, and any combinations thereof.

In some embodiments, the test agent can further comprise a detectablelabel. Examples of a detectable label include, but are not limited to,biotin, a fluorescent dye or molecule, a luminescent or bioluminescentmarker, a radiolabel, an enzyme, a quantum dot, an imaging agent, or anycombination thereof. Methods for detecting various types of thedetectable labels are known in the art. For example, where thedetectable label comprises a fluorescent molecule, signals from theflureoscent labels can be detected, e.g., by fluorescence anisotropyand/or flow cytometry.

In some embodiments, instead of directly detecting a test agent bound tothe modified integrin polypeptide dimer, binding of the test agent tothe modified integrin polypeptide dimer can also be determined by anindirect method, e.g., a competition binding assay. In a competitionbinding assay, the method can further comprise, prior to the detecting,contacting the modified integrin polypeptide dimer with a competingagent.

A competing agent is an agent capable of competing with a test agent tobind the modified integrin polypeptide dimer. Accordingly, a competingagent can be a protein, a peptide, an antibody, a nucleic acid molecule,an apatmer, a peptidomimetic, a small molecule, or any combinationsthereof. In some embodiments, the competing agent can be a competingpeptide. Since the modified integrin polypeptide dimer iscrystallizable, the binding domain of the integrin polypeptide dimer canbe readily identified using any methods known in the art, e.g., X-raycrystallography. Based on the binding domain of the dimer, one candesign a competing agent that can bind to the binding domain. Forexample, an exemplary competing peptide for binding to a modifiedα_(V)β₆ polypeptide dimer described herein comprises an amino acidsequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu (SEQ ID NO: 66), wherein X₁,X₂, and X₃ are each independently an amino acid molecule. Alternatively,another exemplary competing peptide for binding to a modified α_(V)β₆polypeptide dimer described herein comprises an amino acid sequence ofX₃-Arg-Gly-Asp-Leu-X₁-X₂-Ile (SEQ ID NO: 67), wherein X₁, X₂, and X₃ areeach independently an amino acid molecule.

As used herein, the term “amino acid molecule” encompasses a naturallyoccurring amino acid molecule, and a non-naturally occurring amino acidmolecule. One of skill in the art would know that this definitionincludes, D- and L-amino acids; alpha-, beta- and gamma-amino acids;chemically modified amino acids; naturally occurring non-proteinogenicamino acids; rare amino acids; and chemically synthesized compounds thathave properties known in the art to be characteristic of an amino acid.Additionally, each embodiment can include any combinations of thegroups.

The term “naturally occurring amino acid molecule” generally refers toan amino acid molecule that occurs in nature. A naturally occurringamino acid molecule can be a proteinogenic or non-proteinogenic aminoacid. The term “proteinogenic amino acid” as used herein refers to oneof the twenty amino acids used for protein biosynthesis as well as otheramino acids that can be incorporated into proteins during translation(including pyrrolysine and selenocysteine). The twenty proteinogenicamino acids include glycine, alanine, valine, leucine, isoleucine,aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine,arginine, lysine, proline, phenylalanine, tyrosine, tryptophan,cysteine, methionine and histidine. The term “non-proteinogenic aminoacid” as used herein refers to an amino acid that is not encoded by thestandard genetic code, or incorporated into proteins during translation.In some embodiments, the non-proteinogenic amino acid can result fromposttranslational modification of proteins. Exemplary naturallyoccurring non-proteinogenic amino acids include, but are not limited to,hydroxyproline and selenomethionine.

The term “non-naturally occurring amino acid molecule” as used hereinrefers to an amino acid that is not a naturally occurring amino acidmolecule as defined herein. The term “non-naturally occurring amino acidmolecule” can be used synonymously with the term “amino acid analog.” Insome embodiments, the non-naturally occurring amino acid molecule is anamino acid formed by synthetic modification or manipulation of anaturally occurring amino acid. In some embodiments, a non-naturallyoccurring amino acid molecule can be a molecule which departs from thestructure of the naturally occurring amino acids, but which havesubstantially the structure of an amino acid, such that they can besubstituted within a polypeptide which retains its activity, e.g.,ligand-binding activity. Thus, for example, in some embodiments aminoacids can also include amino acids having side chain modifications orsubstitutions, and also include related organic acids, amides or thelike. Examples of non-naturally occurring amino acid molecule include,but are not limited to, homocysteine; phosphoserine; phosphothreonine;phosphotyrosine; γ-carboxyglutamate; hippuric acid;octahydroindole-2-carboxylic acid; statine;1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; penicillamine(3-mercapto-D-valine); ornithine (Orn); citruline; alpha-methyl-alanine;para-benzoylphenylalanine; para-aminophenylalanine;p-fluorophenylalanine; phenylglycine; propargylglycine; N-methylglycins(sarcosine, Sar); and tert-butylglycine; diaminobutyric acid;7-hydroxy-tetrahydroisoquinoline carboxylic acid; naphthylalanine;biphenylalanine; cyclohexylalanine; amino-isobutyric acid (Aib);norvaline; norleucine (Nle); tert-leucine; tetrahydroisoquinolinecarboxylic acid; pipecolic acid; phenylglycine; homophenylalanine;cyclohexylglycine; dehydroleucine; 2,2-diethylglycine;1-amino-1-cyclopentanecarboxylic acid; 1-amino-1-cyclohexanecarboxylicacid; amino-benzoic acid; amino-naphthoic acid; gamma-aminobutyric acid;difluorophenylalanine; nipecotic acid; N-α-imidazole acetic acid (IMA);thienyl-alanine; t-butylglycine; desamino-Tyr; aminovaleric acid (Ava);pyroglutaminic acid (<Glu); α-aminoisobutyric acid (αAib);γ-aminobutyric acid (γAbu); α-aminobutyric acid (αAbu); αγ-aminobutyricacid (αγAbu); 3-pyridylalanine (Pal); Isopropyl-α-N^(ε)lysine (ILys);Napthyalanine (Nal); α-napthyalanine (α-Nal); β-napthyalanine (β-Nal);Acetyl-β-napthyalanine (Ac-β-napthyalanine); α, β-napthyalanine;N^(ε)-picoloyl-lysine (PicLys); 4-halo-Phenyl; 4-pyrolidylalanine;isonipecotic carboxylic acid (inip); beta-amino acids; and isomers,analogs and derivatives thereof.

In some embodiments, a non-naturally occurring amino acid molecule canbe a chemically modified amino acid. As used herein, the term“chemically modified amino acid” refers to an amino acid that has beentreated with one or more reagents.

In some embodiments, a non-naturally occurring amino acid molecule canbe a beta-amino acid. Exemplary beta-amino acids include, but are notlimited to, L-β-Homoproline hydrochloride;(±)-3-(Boc-amino)-4-(4-biphenylyl)butyric acid;(±)-3-(Fmoc-amino)-2-phenylpropionic acid;(1S,3R)-(+)-3-(Boc-amino)cyclopentanecarboxylic acid;(2R,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid;(2S,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid;(R)-2-[(Boc-amino)methyl]-3-phenylpropionic acid;(R)-3-(Boc-amino)-2-methylpropionic acid;(R)-3-(Boc-amino)-2-phenylpropionic acid;(R)-3-(Boc-amino)-4-(2-naphthyl)butyric acid;(R)-3-(Boc-amino)-5-phenylpentanoic acid;(R)-3-(Fmoc-amino)-4-(2-naphthyl)butyric acid;(R)-(−)-Pyrrolidine-3-carboxylic acid; (R)-Boc-3,4-dimethoxy-β-Phe-OH;(R)-Boc-3-(3-pyridyl)-β-Ala-OH; (R)-Boc-3-(trifluoromethyl)-β-Phe-OH;(R)-Boc-3-cyano-β-Phe-OH; (R)-Boc-3-methoxy-β-Phe-OH;(R)-Boc-3-methyl-β-Phe-OH; (R)-Boc-4-(4-pyridyl)-β-Homoala-OH;(R)-Boc-4-(trifluoromethyl)-β-Homophe-OH;(R)-Boc-4-(trifluoromethyl)-β-Phe-OH; (R)-Boc-4-bromo-β-Phe-OH;(R)-Boc-4-chloro-β-Homophe-OH; (R)-Boc-4-chloro-β-Phe-OH;(R)-Boc-4-cyano-β-Homophe-OH; (R)-Boc-4-cyano-β-Phe-OH;(R)-Boc-4-fluoro-β-Phe-OH; (R)-Boc-4-methoxy-β-Phe-OH;(R)-Boc-4-methyl-β-Phe-OH; (R)-Boc-β-Tyr-OH;(R)-Fmoc-4-(3-pyridyl)-β-Homoala-OH; (R)-Fmoc-4-fluoro-β-Homophe-OH;(S)-(+)-Pyrrolidine-3-carboxylic acid;(S)-3-(Boc-amino)-2-methylpropionic acid;(S)-3-(Boc-amino)-4-(2-naphthyl)butyric acid;(S)-3-(Boc-amino)-5-phenylpentanoic acid;(S)-3-(Fmoc-amino)-2-methylpropionic acid;(S)-3-(Fmoc-amino)-4-(2-naphthyl)butyric acid;(S)-3-(Fmoc-amino)-5-hexenoic acid;(S)-3-(Fmoc-amino)-5-phenyl-pentanoic acid;(S)-3-(Fmoc-amino)-6-phenyl-5-hexenoic acid;(S)-Boc-2-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-2-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-2-(trifluoromethyl)-β-Phe-OH; (S)-Boc-2-cyano-β-Homophe-OH;(S)-Boc-2-methyl-β-Phe-OH; (S)-Boc-3,4-dimethoxy-β-Phe-OH;(S)-Boc-3-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-3-(trifluoromethyl)-β-Phe-OH; (S)-Boc-3-methoxy-β-Phe-OH;(S)-Boc-3-methyl-β-Phe-OH; (S)-Boc-4-(4-pyridyl)-β-Homoala-OH;(S)-Boc-4-(trifluoromethyl)-β-Phe-OH; (S)-Boc-4-bromo-β-Phe-OH;(S)-Boc-4-chloro-β-Homophe-OH; (S)-Boc-4-chloro-β-Phe-OH;(S)-Boc-4-cyano-β-Homophe-OH; (S)-Boc-4-cyano-β-Phe-OH;(S)-Boc-4-fluoro-β-Phe-OH; (S)-Boc-4-iodo-β-Homophe-OH;(S)-Boc-4-methyl-β-Homophe-OH; (S)-Boc-4-methyl-β-Phe-OH;(S)-Boc-β-Tyr-OH; (S)-Boc-γ, γ-diphenyl-β-Homoala-OH;(S)-Fmoc-2-methyl-β-Homophe-OH; (S)-Fmoc-3,4-difluoro-β-Homophe-OH;(S)-Fmoc-3-(trifluoromethyl)-β-Homophe-OH;(S)-Fmoc-3-cyano-β-Homophe-OH; (S)-Fmoc-3-methyl-β-Homophe-OH;(S)-Fmoc-γ,γ-diphenyl-β-Homoala-OH; 2-(Boc-aminomethyl)phenylaceticacid; 3-Amino-3-(3-bromophenyl)propionic acid;3-Amino-4,4,4-trifluorobutyric acid; 3-Aminobutanoic acid;DL-3-Aminoisobutyric acid; DL-β-Aminoisobutyric acid puriss;DL-β-Homoleucine; DL-β-Homomethionine; DL-β-Homophenylalanine;DL-β-Leucine; DL-β-Phenylalanine; L-β-Homoalanine hydrochloride;L-β-Homoglutamic acid hydrochloride; L-β-Homoglutamine hydrochloride;L-β-Homohydroxyproline hydrochloride; L-β-Homoisoleucine hydrochloride;L-β-Homoleucine hydrochloride; L-β-Homolysine dihydrochloride;L-β-Homomethionine hydrochloride; L-β-Homophenylalanine allyl esterhydrochloride; L-β-Homophenylalanine hydrochloride; L-β-Homoserine;L-β-Homothreonine; L-β-Homotryptophan hydrochloride; L-β-Homotyrosinehydrochloride; L-β-Leucine hydrochloride; Boc-D-β-Leu-OH;Boc-D-β-Phe-OH; Boc-β³-Homopro-OH; Boc-β-Glu(OBzl)-OH;Boc-β-Homoarg(Tos)-OH; Boc-β-Homoglu(OBzl)-OH; Boc-β-Homohyp(Bzl)-OH(dicyclohexylammonium) salt technical; Boc-β-Homolys(Z)—OH;Boc-β-Homoser(Bzl)-OH; Boc-β-Homothr(Bzl)-OH; Boc-β-Homotyr(Bzl)-OH;Boc-β-Ala-OH; Boc-β-Gln-OH; Boc-β-Homoala-OAll; Boc-β-Homoala-OH;Boc-β-Homogln-OH; Boc-β-Homoile-OH; Boc-β-Homoleu-OH; Boc-β-Homomet-OH;Boc-β-Homophe-OH; Boc-β-Homotrp-OH; Boc-β-Homotrp-OMe; Boc-β-Leu-OH;Boc-β-Lys(Z)—OH (dicyclohexylammonium) salt; Boc-β-Phe-OH; Ethyl3-(benzylamino)propionate; Fmoc-D-β-Homophe-OH; Fmoc-L-β³-homoproline;Fmoc-β-D-Phe-OH; Fmoc-β-Gln(Trt)-OH; Fmoc-β-Glu(OtBu)—OH;Fmoc-β-Homoarg(Pmc)-OH; Fmoc-β-Homogln(Trt)-OH; Fmoc-β-Homoglu(OtBu)—OH;Fmoc-β-Homohyp(tBu)—OH; Fmoc-β-Homolys(Boc)-OH; Fmoc-β-Homoser(tBu)—OH;Fmoc-β-Homothr(tBu)—OH; Fmoc-β-Homotyr(tBu)—OH; Fmoc-β-Ala-OH;Fmoc-β-Gln-OH; Fmoc-β-Homoala-OH; Fmoc-β-Homogln-OH; Fmoc-β-Homoile-OH;Fmoc-β-Homoleu-OH; Fmoc-β-Homomet-OH; Fmoc-β-Homophe-OH;Fmoc-β-Homotrp-OH; Fmoc-β-Leu-OH; Fmoc-β-Phe-OH;N-Acetyl-DL-β-phenylalanine; Z-D-β-Dab(Boc)-OH; Z-D-β-Dab(Fmoc)-OHpurum; Z-DL-β-Homoalanine; Z-β-D-Homoala-OH; Z-β-Glu(OtBu)—OH technical;Z-β-Homotrp(Boc)-OH; Z-β-Ala-OH purum; Z-β-Ala-ONp purum;Z-β-Dab(Boc)-OH; Z-β-Dab(Fmoc)-OH; Z-β-Homoala-OH; β-Alanine; β-AlanineBioXtra; β-Alanine ethyl ester hydrochloride; β-Alanine methyl esterhydrochloride; β-Glutamic acid hydrochloride;cis-2-Amino-3-cyclopentene-1-carboxylic acid hydrochloride;cis-3-(Boc-amino)cyclohexanecarboxylic acid; andcis-3-(Fmoc-amino)cyclohexanecarboxylic acid.

In some embodiments, the X₁ can be a Gly molecule or an analog thereof.

In some embodiments, the X₂ can be an Arg molecule or an analog thereof.

In some embodiments, the X₃ can be a Gly molecule or an analog thereof.

In some embodiments, a competing peptide for binding to a modifiedα_(V)β₆ polypeptide dimer described herein can comprise an amino acidsequence of Gly-Arg-Gly-Asp-Leu-Gly-Arg-Leu (SEQ ID NO: 68). In someembodiments, a competing peptide for binding to a modified α_(V)β₆polypeptide dimer described herein can comprise an amino acid sequenceof Gly-Arg-Gly-Asp-Leu-Gly-Arg-Ile (SEQ ID NO: 69).

In some embodiments, the competing agent can further comprise adetectable label. As used herein in reference to a test agent or acompeting agent, the term “detectable label” refers to a compositioncapable of producing a detectable signal indicative of the presence of atarget with the detectable label attached thereto. Detectable labelsinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Suitable labels include fluorescent molecules, radioisotopes, nucleotidechromophores, enzymes, substrates, chemiluminescent moieties,bioluminescent moieties, and the like. As such, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means needed for themethods and devices described herein.

A wide variety of fluorescent reporter dyes are known in the art.Typically, the fluorophore is an aromatic or heteroaromatic compound andcan be a pyrene, anthracene, naphthalene, acridine, stilbene, indole,benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine,salicylate, anthranilate, coumarin, fluorescein, rhodamine or other likecompound.

Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™;Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™;Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647T^(M); Alexa Fluor660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate;APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; AstrazonRed 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ;Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); BG-647; Bimane;Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550;Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl;Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; BodipyTMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-XSE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; CalceinBlue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca2+ Dye; CalciumGreen-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; CalciumOrange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™;Cascade Yellow; Catecholamine; CFDA; CFP-Cyan Fluorescent Protein;Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine;Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazineh; Coelenterazine hcp; Coelenterazine ip; Coelenterazine O; CoumarinPhalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2;Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; DansylDHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS;Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1);Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4;Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UVexcitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65 Å; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary detectable labels include luminescent and bioluminescentmarkers (e.g., biotin, luciferase (e.g., bacterial, firefly, clickbeetle and the like), luciferin, and aequorin), radiolabels (e.g., 3H,125I, 35S, 14C, or 32P), enzymes (e.g., galactosidases, glucorinidases,phosphatases (e.g., alkaline phosphatase), peroxidases (e.g.,horseradish peroxidase), and cholinesterases), and calorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,4,277,437, 4,275,149, and 4,366,241, each of which is incorporatedherein by reference.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels can be detected using photographicfilm or scintillation counters, fluorescent markers can be detectedusing a photo-detector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with an enzyme substrate anddetecting the reaction product produced by the action of the enzyme onthe enzyme substrate, and calorimetric labels can be detected byvisualizing the colored label.

In some embodiments, the detectable label is a fluorophore or a quantumdot. Without wishing to be bound by a theory, using a fluorescentreagent can reduce signal-to-noise in the imaging/readout, thusmaintaining sensitivity.

In some embodiments, a detectable label can include, but is not limitedto, biotin, a fluorescent dye or molecule, a luminescent orbioluminescent marker, a radiolabel, an enzyme, a quantum dot, animaging agent, or any combination thereof. Methods for detecting varioustypes of the detectable labels are known in the art and are describedabove.

In some embodiments where the detectable label comprises a fluorescentmolecule, signals from the flureoscent labels can be detected, e.g., byfluorescence anisotropy and/or flow cytometry. Fluorescence anisotropycan be used to measure the binding constants and kinetics of reactionsthat cause a change in the rotational time of the molecules. If thefluorophore (detectable label) remains bound to a ligand (e.g., a testagent or a competing agent described below) without binding to integrin,the rate at which it tumbles can decrease significantly when it is boundtightly to a large protein such as an integrin. If the fluorophore(detectable label) conjugated to a test agent and/or competing agentwhich binds to the larger integrin protein in a binding pair, thedifference in polarization between bound and unbound states will besmaller (because the unbound protein will already be fairly stable andtumble slowly to begin with) and the measurement will be less accurate.The degree of binding is calculated by using the difference inanisotropy of the partially bound, free and fully bound (large excess ofprotein) states measured by titrating the two binding partners. Methodsfor using fluorescence anisotropy to identify agents with affinity for atarget molecule are known in the art, including, e.g., the methods asdescribed in the International Application No. WO 19998/039484, thecontent of which is incorporated herein by reference.

Where the competing agent comprises a detectable label, signal from thecompeting agent is detected instead of the test agent. Thus, if thesignal from the competing agent is reduced upon contacting the modifiedintegrin polypeptide dimer with the test agent, where the concentrationsof the test agent and the competing agent are the same, this indicatesthat the test agent has a higher binding affinity than the competingagent to the modified integrin polypeptide dimer described herein.

Accordingly, yet another aspect provided herein relates to a method fordetermining binding affinity of a test agent to an integrin heterodimer.The method comprises (i) contacting one or more modified integrinpolypeptide dimers described herein with a test agent and a competingagent, wherein the competing agent comprises a detectable label and iscapable of competing with the test agent to bind the modified integrinpolypeptide dimer; and (ii) detecting a signal from the detectable labelof the competing agent that forms a complex with the integrin. Adecrease in the detected signal (e.g., by at least about 10% or more,including, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95% or higher) relative toa signal corresponding to saturation binding of the competing agent tothe modified integrin polypeptide dimer indicates that the test agenthas a higher binding affinity than the competing agent to the integrin.

The term “saturation binding” generally refers to all binding sites of atarget molecule being occupied by interaction with one specific ligand.Therefore, as used herein, the term “saturation binding” refers to allbinding sites of the modified integrin polypeptide dimers describedherein being occupied by a competing agent in the absence of any testagent. However, upon addition of a test agent that has a higher bindingaffinity than the competing agent to the modified integrin polypeptidedimers described herein, fewer molecules of competing agent can bind tothe modeified polypeptide dimers, resulting in a reduced signal from thecompeting agent.

As used herein, the term “binding affinity” generally refers to anoverall binding property of a first agent (e.g., a ligand or a testagent) interacting with a second agent (e.g., a target protein such asintegrin α_(V)β₆ heterodimer) under a specific condition, and theoverall binding property is typically dependent on intrinsiccharacteristics of the first agent and the second agent including, butnot limited to, the conformation of the first agent and/or the secondagent, single-bond affinity, avidity, as well as the surrounding/ambientcondition for the binding interaction, e.g., but not limited to,concentration of the first agent and/or the second agent, and/or thepresence of other interfering molecules during the binding interactionbetween the first and the second agents. Different measures of a bindingaffinity of an agent are known in the art. In some embodiments, thebinding affinity of a first agent for a second agent can be indicated bydissociation constant (K_(d)) for binding of the first agent to thesecond agent. The dissociation constant (K_(d)) is an equilibriumconstant that generally measures the propensity of a bound complex toseparate (dissociate) reversibly into separate agents. In theseembodiments, a higher dissociation constant indicates a lower effectivebinding affinity. Alternatively, the effective binding affinity of afirst agent for a second agent can be indicated by an associationconstant (K) for binding of the first agent to the second agent. Theassociation constant (K) is the inverse of the dissociation constant(K_(d)), i.e., a higher association constant indicates a highereffective binding affinity.

As used herein, the term “single-bond affinity” refers to the strengthof a single bond interaction, including but not limited to hydrogenbonds, electrostatic bonds, van der Waals forces, hydrophobic forces, orany combinations thereof.

As used herein, the term “avidity” refers to the combined strength ofmultiple bond interactions. Avidity is distinct from affinity orsingle-bond affinity, which is the strength of a single bondinteraction. In general, avidity is the combined synergistic strength ofbond affinities rather than the sum of bonds. Accordingly, avidity isgenerally used to describe one agent having multiple interactions withanother agent. For example, a ligand or a test agent can have multipleinteractions with a modified integrin polypeptide dimer (e.g., amodified integrin α_(v)β₆ polypeptide dimer described herein).

Drug Discovery and Development Process

As noted above, the modified integrin polypeptide dimers describedherein can form crystal structures. Thus, the binding domain of thedimer can be readily identified using any methods known in the art,e.g., X-ray crystallography. Indeed, the inventors have identified anovel hydrophobic binding pocket of an integrin α_(V)β₆ heterodimerbased on the crystal structure of the modified integrin α_(V)β₆polypeptide dimer described herein. Accordingly, the inventors employthe information of the novel hydrophobic binding pocket to design apharmacophore model for an agent that can bind to the hydrophobicbinding pocket of the integrin α_(V)β₆ heterodimer.

In some embodiments, the pharmacophore model can be designed for ananti-α_(V)β₆ inhibitor. Therefore, provided herein is also a method ofidentifying an anti-α_(V)β₆ inhibitor. The method comprises: (a)generating on a computer a molecular representation of a pharmacophorecomprising a basic functional group, an acidic functional group forcoordination of a metal ion to a metal ion-dependent adhesion site(MIDAS) in integrin β₆ polypeptide, a first hydrophobic functionalgroup, and a second hydrophobic functional group, wherein the functionalgroups are arranged to satisfy the following conditions:

the distance between the first hydrophobic functional group (H1) and thesecond hydrophobic functional group (H2) is about 7-8 Å; the distancebetween the second hydrophobic functional group (H2) and the basicfunctional group (B) is about 8-9 Å; the distance between the basicfunctional group (B) and the acidic functional group (A) is about 15-16Å; the distance between the first hydrophobic functional group (H1) andthe acidic functional group (A) is about 14.5-15.5 Å; and the distancebetween the second hydrophobic functional group (H2) and the acidicfunctional group (A) is about 19-20 Å; and

the angle formed by H1-A-B is about 20°-24°; the angle formed by H1-A-H2is about 17°-21°; the angle formed by H2-A-B is about 26°-30°; the angleformed by A-B-H1 is about 68°-72°; the angle formed by A-B-H2 is about96°-100°; and the angle formed by H1-B-H2 is about 49°-53°;

(b) generating on a computer atomic coordinates of an α_(V)β₆ integrinprotein or a portion thereof having at least a hydrophobic bindingpocket in β₆ subunit; and (c) determining on a computer likelihood ofthe molecular representation interacting with one or more residues ofthe computer-generated α_(V)β₆ integrin protein or a portion thereof,thereby identifying a candidate anti-α_(V)β₆ inhibitor.

In some embodiments, the first and second hydrophobic functional groupscan each independently have an aromatic ring (aryl) or linear moiety.

In some embodiments, the functional groups of the pharmacophore can bearranged to satisfy the following conditions:

the distance between the first hydrophobic functional group and thesecond hydrophobic functional group is about 7.403 Å; the distancebetween the second hydrophobic functional group and the basic functionalgroup is about 8.462 Å; the distance between the basic functional groupand the acidic functional group is about 15.639 Å; the distance betweenthe first hydrophobic functional group and the acidic functional groupis about 15.005 Å; and the distance between the second hydrophobicfunctional group and the acidic functional group is about 19.553 Å; and

the angle formed by H1-A-B is about 22.4°; the angle formed by H1-A-H2is about 19.4°; the angle formed by H2-A-B is about 28.7°; the angleformed by A-B-H1 is about 70.7°; the angle formed by A-B-H2 is about98.1°; and the angle formed by H1-B-H2 is about 51.1°.

In some embodiments, the determining step (c) can further comprisefitting the molecular representation to determine the probability of themolecular representation interacting with one or more residues of thecomputer generated α_(V)β₆ integrin protein or a portion thereof. By“fitting” is meant determining by automatic, or semi-automatic means,interactions between one or more atoms of a candidate molecule and atleast one atom of a α_(V)β₆ integrin protein or a portion thereofstructure and calculating the extent to which such interactions arestable. Interactions include attraction and repulsion, brought about bycharge, and/or steric considerations and the like. Interactions of thistype can be modeled computationally. An example of such computationwould be via a force field such as Amber (Cornell et al. A SecondGeneration Force Field for the Simulation of Proteins, Nucleic Acids,and Organic Molecules, Journal of the American Chemical Society, (1995),117(19), 5179-97) which would assign partial charges to atoms on theprotein and ligand and evaluate the electrostatic interaction energybetween a protein and ligand atom using the Coulomb potential. The Amberforce field would also assign van der Waals energy terms to assess theattractive and repulsive steric interactions between two atoms. Othermethods of assessing interactions are available and would be known toone skilled in the art of designing molecules consistent with specifiedpharmacophores.

As used herein, the term “pharmacophore” refers to a best descriptionfor a three-dimensional orientation of a set of features which describethe physical, chemical, steric, and/or electronic environment of theactive sites of a molecule, compound, or a structure, said featurescomprising. Examples of pharmacophoric features include, but are notlimited to, the hydrogen bond donor feature, the hydrogen bond acceptorfeature, the hydrophobic or lipophilic region feature, the ionizableregion feature (e.g., acidic or basic functional groups), and the ringaromatic feature. A pharmacophore model generally explains howstructurally diverse molecules can bind to a common target site.Furthermore, pharmacophore models can be used to identify through denovodesign or virtual screening novel ligands that will bind to the sametarget site.

As used herein, the term “active site” refers to a site (such as anatom, a functional group of an amino acid residue or a plurality of suchatoms and/or groups) that is capable of binding to an integrin αβheterodimer and block the corresponding integrin signaling.

The term “hydrogen bond” as used herein refers to an interaction thatoccurs whenever a suitable donor atom bearing a proton, H, and asuitable acceptor atom. Sometimes, a single acceptor atom can form aplurality of hydrogen bonds with a plurality of protons on suitabledonor atoms. Sometimes, a single proton on a donor atom can formhydrogen bonds with a plurality of suitable acceptor atoms. For example,the proton on a —NH-group may form a separate hydrogen bond with each ofthe two oxygen atoms in a carboxylate anion. Suitable donor and acceptoratoms are well understood in medicinal chemistry (G. C. Pimentel and A.L. McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960; R. Taylorand O. Kennard, Hydrogen Bond Geometry in Organic Crystals, Accounts ofChemical Research, 17, pp. 320-326 (1984)).

As used herein, the term “hydrogen bond donor” refers to a chemicalstructure containing a suitable hydrogen bond donor atom bearing one ormore protons. It refers to a group having a hydrogen atom capable offorming a hydrogen bond with acceptor atom in the same or an adjacentmolecule; see for example “Advanced Organic Chemistry” by Jerry March,4th edition, which is incorporated herein by reference. Examples ofdonor atoms having one proton are —NH, C—NH2, C—NH, C—OH, C—SH, oraromatic C—H. Examples of donor atoms having more than one proton are—NH2, —[NH3]+ and —[NH4]+.

As used herein, the term “hydrogen bond acceptor” refers to a chemicalstructure containing a suitable hydrogen bond acceptor atom. Itgenerally refers to a group capable of forming a hydrogen bond with ahydrogen atom in the same or an adjacent molecule; see for example“Advanced Organic Chemistry” by Jerry March, 4th edition, which isincorporated herein by reference. Examples of acceptor atoms includefluorine, oxygen, sulfur and nitrogen and thus in the present context,hydrogen bond acceptors include nitrogen, oxygen and sulphur atoms; andgroups containing nitrogen, oxygen and sulphur atoms.

The term “lipophilic” or “hydrophobic” refers to a non-polar moiety thattends not to dissolve in water and is fat-soluble. Hydrophobic moietiesinclude, but are not limited to, hydrocarbons, such as alkanes, alkenes,alkynes, cycloalkanes, ethers, cycloalkenes, cycloalkynes and aromaticcompounds, such as aryls, certain saturated and unsaturatedheterocycles, and moieties that are substantially similar to the sidechains of lipophilic natural and unnatural amino acids, includingvaline, leucine, isoleucine, methionine, phenylalanine, α-aminoisobutyric acid, alloisoleucine, tyrosine, and tryptophan. Lipophilicinteractions can be modeled using a variety of means. For example theChemScore function (Eldridge M D; Murray C W; Auton T R; Paolini G V;Mee R P Empirical scoring functions: I. The development of a fastempirical scoring function to estimate the binding affinity of ligandsin receptor complexes, Journal of computer-aided molecular design (1997September), 11(5), 425-45) assigns protein and ligand atoms ashydrophobic or polar, and a favorable energy term is specified for theinteraction between two hydrophobic atoms. Other methods of assessingthe hydrophobic contributions to ligand binding are available and thesewould be known to one skilled in the art.

The term “polar” as used herein refers to compounds having one or morepolar bonds in which the electron density of the bond lies closer to oneatom than the other as one of the atoms is more electronegative than theother. This means that one of the atoms develops a degree of positivecharge while the other some degree of negative charge. Compounds havingpolar bonds generally have dipole moments. In contrast, covalent bondsbetween atoms having the same electronegativity are symmetric.

As used herein, the term “acidic” refers to the tendency of compounds todonate a proton (H+) (Bronsted-Lowry Theory) or accept an electron pairinto an empty orbital (Lewis Theory of Acids and Bases). An exemplaryacidic functional group is a carboxylic acid group.

As used herein, the term “basic” refers to the tendency of compounds toaccept a proton (H+) (Bronsted-Lowry Theory) or donate an electron pair(Lewis Theory of Acids and Bases). An exemplary basic functional groupis an amino group.

In accordance with some aspects described herein, a phamacophore refersto a best description for a three-dimensional orientation of a set offeatures, which describe the physical, chemical, steric, and/orelectronic environment of the active sites of an anti-α_(V)β₆ integrininhibitor. In some embodiments, the pharmacophore model for ananti-α_(V)β₆ inhibitor can comprise a basic functional group, an acidicfunctional group for coordination of a metal ion to a metalion-dependent adhesion site (MIDAS) in integrin β₆ polypeptide, a firsthydrophobic functional group, and a second hydrophobic functional group.

Determination of an anti-α_(V)β₆ inhibitor pharmacophore can greatlyassist the process of rational drug design. This information can be usedfor rational design of anti-α_(V)β₆ inhibitors, e.g. by computationaltechniques which identify possible binding ligands for the bindingsites, by linked-fragment approaches to drug design, and bystructure-based design based on the location of bound ligand. Thesetechniques are discussed in more detail below.

Greer et al. (J. of Medicinal Chemistry, Vol. 37, (1994), 1035-1054)describe an iterative approach to ligand design based on repeatedsequences of computer modelling, protein-ligand complex formation andX-ray crystallographic or NMR spectroscopic analysis. Thus novelthymidylate synthase inhibitor series were designed de novo by Greer etal., and anti-α_(V)β₆ inhibitors may also be designed in a similar way.More specifically, using e.g. GRID on the solved 3D structure of theintegrin α_(V)β₆ heterodimer, a ligand (e.g. a candidate modulator inparticular an inhibitor) for integrin α_(V)β₆ heterodimer can bedesigned that complements the functionalities of integrin α_(V)β₆binding sites. The ligand can then be synthesised, formed into a complexwith the integrin α_(V)β₆, and the complex then analysed by X-raycrystallography to identify the actual position of the bound ligand. Thestructure and/or functional groups of the ligand can then be adjusted,if necessary, in view of the results of the X-ray analysis, and thesynthesis and analysis sequence repeated until an optimised ligand isobtained. Related approaches to structure-based drug design are alsodiscussed in Bohacek et al., Medicinal Research Reviews, Vol. 16,(1996), 3-50.

Structure-based drug design and in silico approaches to drug designrequire accurate information on the atomic coordinates of targetproteins or receptors. The coordinates used in the design, selection andanalysis of the candidate anti-α_(V)β₆ inhibitors can be crystalstructures for examples obtained from the Protein Data Bank or obtainedin house, or homology models. In some embodiments, the coordinates usedin the design, selection and analysis of the candidate anti-α_(V)β₆inhibitors can be crystal structure of α_(V)β₆ headpiece identified bythe inventors as described in the Examples. Homology models can begenerated using “homology modelling.” By “homology modelling”, it ismeant the prediction of structures for example of integrin α_(V)β₆,based either on X-ray crystallographic data (for example of α_(V)β₆headpiece) or computer-assisted de novo prediction of structure, basedupon manipulation of the coordinate data of existing integrin domainstructures. Homology modelling as such is a technique that is well knownto those skilled in the art (see e.g. Greer, Science, Vol. 228, (1985),1055, and Blundell et al., Eur. J. Biochem, Vol. 172, (1988), 513). Thetechniques described in these references, as well as other homologymodeling techniques, generally available in the art, may be used inperforming the present invention.

Homology modelling comprises the steps of: (a) aligning a representationof an amino acid sequence of a target protein of unknownthree-dimensional structure with the amino acid sequence of the knownprotein to match homologous regions of the amino acid sequences; (b)modeling the structure of the matched homologous regions of said targetprotein of unknown structure on the corresponding regions of the knownstructure; and (c) determining a conformation for said target protein ofunknown structure which substantially preserves the structure of saidmatched homologous regions. In particular one or all of steps (a) to (c)are performed by computer modeling.

The term “homologous regions” describes amino acid residues in twosequences that are identical or have similar (e.g. aliphatic, aromatic,polar, negatively charged, or positively charged) side-chain chemicalgroups. Identical and similar residues in homologous regions aresometimes described as being respectively “invariant” and “conserved” bythose skilled in the art.

In general, the method involves comparing the amino acid sequence ofproteins of unknown structure with the proteins of known structure byaligning the amino acid sequences (Dunbrack et al., Folding and Design,2, (1997), 27-42). Amino acids in the sequences are then compared andgroups of amino acids that are homologous (conveniently referred to as“corresponding regions”) are grouped together. This method detectsconserved regions of the polypeptides and accounts for amino acidinsertions or deletions. Homology between amino acid sequences can bedetermined using commercially available algorithms. The programs BLAST,gapped BLAST, BLASTN, PSI-BLAST and BLAST 2 sequences (provided by theNational Center for Biotechnology Information) are widely used in theart for this purpose, and can align homologous regions of two amino acidsequences.

Once the amino acid sequences of the polypeptides with known and unknownstructures are aligned, the structures of the conserved amino acids in acomputer representation of the polypeptide with known structure aretransferred to the corresponding amino acids of the polypeptide whosestructure is unknown. For example, a tyrosine in the amino acid sequenceof known structure may be replaced by a phenylalanine, the correspondinghomologous amino acid in the amino acid sequence of unknown structure.

The structures of amino acids located in non-conserved regions can beassigned manually by using standard peptide geometries or by molecularsimulation techniques, such as molecular dynamics. The final step in theprocess is accomplished by refining the entire structure using moleculardynamics and/or energy minimization.

Linked-fragment approaches to drug design can also be used to designanti-α_(V) β₆ inhibitors comprising the pharmacophore. Thefragment-linking approach involves determining (computationally orexperimentally) the binding locations of plural ligands to a targetmolecule, and then constructing a molecular scaffold to connect theligands together in such a way that their relative binding positions arepreserved. The ligands may be provided computationally and modeled in acomputer system, or provided in an experimental setting, wherein, forexample, X-ray crystallography is used to determine their location. Thepharmacophore of anti-α_(V) β₆ inhibitor described herein can beconsidered to be one such fragment for use in a linked fragmentapproach.

The binding site of two or more ligands are determined and may beconnected to form a potential lead compound that can be further refinedusing e.g. the iterative technique of Greer et al. For a virtuallinked-fragment approach see Verlinde et al., J. of Computer-AidedMolecular Design, 6, (1992), 131-147, and for NMR and X-ray approachessee Shuker et al., Science, 274, (1996), 1531-1534 and Stout et al.,Structure, 6, (1998), 839-848. The use of these approaches with thepharmacophore described herein can be used to design anti-α_(V) β₆inhibitors.

Many of the techniques and approaches to structure-based drug designdescribed above rely at some stage on X-ray analysis to identify thebinding position of a ligand in a ligand-protein complex. A common wayof doing this is to perform X-ray crystallography on the complex,produce a difference Fourier electron density map, and associate aparticular pattern of electron density with the ligand. However, inorder to produce the map (as explained e.g. by Blundell, T L andJohnson, L N, in Protein Crystallography, Academic Press, New York,London and San Francisco, (1976)) it is necessary to know beforehand theprotein 3D structure (or at least the protein structure factors).

The provision of the pharmacophore described herein will also allow thedevelopment of compounds or molecules which interact with the bindingpocket regions of integrin α_(V)β₆ heterodimer (for example to act asinhibitors of an α_(V)β₆ integrin) based on a fragment linking orfragment growing approaches.

For example, pharmacophore model for an anti-α_(V)β₆ inhibitor describedherein can provide a starting point for medicinal chemistry to optimizethe interactions using a structure-based approach. The fragments can becombined onto a template e.g., the pharmacophore model for ananti-α_(V)β₆ inhibitor described herein could be used as the startingpoint for ‘growing out’ an inhibitor into other pockets of the protein(Blundell T L, Jhoti H, Abell C, Nature Reviews Drug Discovery, 1,45-54, 2002, Carr, R; Jhoti, H; Drug Discov. Today, 2002, 7(9),522-527). The fragments can be positioned in the binding pockets ofintegrin α_(V)β₆ and then ‘grown’ to fill the space available, exploringthe electrostatic, van der Waals or hydrogen-bonding interactions thatare involved in molecular recognition. The potency of the originalweakly binding fragment thus can be rapidly improved using iterativestructure-based chemical synthesis.

At one or more stages in the fragment growing approach, the compound maybe synthesized and tested in a biological system for its activity. Thiscan be used to guide the further growing out of the fragment.

Where two fragment-binding regions are identified, a linked fragmentapproach may be based upon attempting to link the two fragmentsdirectly, or growing one or both fragments in the manner described abovein order to obtain a larger, linked structure, which may have thedesired properties.

In some embodiments, the pharmacophore model for an anti-α_(V)β₆inhibitor described herein can be used in in silico analysis and design.Current computational techniques provide a powerful alternative to theneed to generate crystals and generate and analyse diffraction data.Accordingly, one aspect described herein relates to in silico methodsdirected to the analysis and development of anti-α_(V)β₆ inhibitorscomprising the pharmacophoric feature described herein, or derived ordesigned from the molecular fragments herein.

The approaches to structure-based drug design described below allrequire initial identification of possible compounds for interactionwith the target bio-molecule (in this case integrin α_(V)β₆). Sometimesthese compounds are known e.g., from the research literature. However,when they are not, or when novel compounds are wanted, a first stage ofthe drug design program may involve computer-based in silico screeningof compound databases (such as the Cambridge Structural Database or theAvailable Chemical Directory (ACD) (MDL Information Systems, SanLeandro, Calif., USA) with the aim of identifying compounds whichinteract with the binding site or sites of the target bio-molecule.Screening selection criteria can be based on pharmacokinetic propertiessuch as metabolic stability and toxicity or the pharmacophore of theinvention. The pharmacophore can thus be used as selection criteria orfilter for database screening.

Thus, as a result of the determination of the integrin α_(V)β₆selectivity pharmacophore more purely computational techniques forrational drug design may also be used to design anti-α_(V)β₆ selectiveinhibitors (for an overview of these techniques see e.g. Walters et al,Drug Discovery Today, Vol. 3, No. 4, (1998), 160-178; Abagyan, R.;Totrov, M. Curr. Opin. Chem. Biol. 2001, 5, 375-382). For example,automated ligand-receptor docking programs (discussed e.g. by Jones etal. in Current Opinion in Biotechnology, Vol. 6, (1995), 652-656 andHalperin, I.; Ma, B.; Wolfson, H.; Nussinov, R. Proteins 2002, 47,409-443), can be used to design potential anti-α_(V)β₆ inhibitors on thebasis of the pharmacophore described herein.

The determination of the pharmacophore for anti-α_(V) β₆ selectiveinhibitors provides a basis for the design of new and specific ligandsfor integrin α_(V) β₆. For example, computer modeling programs may beused to design different molecules expected to interact with bindingcavities or other structural or functional features of integrin α_(V)β₆. Examples of this are discussed in Schneider, G.; Bohm, H. J. DrugDiscov. Today 2002, 7, 64-70.

More specifically, the interaction of a compound with integrin α_(V)β₆can be examined through the use of computer modeling using a dockingprogram such as GOLD (Jones et al., J. Mol. Biol., 245, 43-53 (1995),Jones et al., J. Mol. Biol., 267, 727-748 (1997)), GRAMM (Vakser, I. A.,Proteins, Suppl., 1:226-230 (1997)), DOCK (Kuntz et al, J. Mol. Biol.1982, 161, 269-288, Makino et al, J. Comput. Chem. 1997, 18, 1812-1825),AUTODOCK (Goodsell et al, Proteins 1990, 8, 195-202, Morris et al, J.Comput. Chem. 1998, 19, 1639-1662.), FlexX, (Rarey et al, J. Mol. Biol.1996, 261, 470-489) or ICM (Abagyan et al, J. Comput. Chem. 1994, 15,488-506). This procedure can include computer fitting of compounds tointegrin α_(V)β₆ to ascertain how well the shape and the chemicalstructure of the compound will bind to integrin α_(V)β₆. In addition,the pharmacophore of an anti-α_(V)β₆ inhibitor described can allow thegeneration of highly predictive pharmacophore models for virtual libraryscreening or compound design.

Also, computer-assisted, manual examination of the binding sitestructure of α_(V) β₆ can be performed. The use of programs such as GRID(Goodford, J. Med. Chem., 28, (1985), 849-857)—a program that determinesprobable interaction sites between molecules with various functionalgroups and an enzyme surface-may also be used to analyse the bindingcavity or cavities to predict partial structures of inhibitingcompounds.

Computer programs (e.g. molecular simulation methods such as Tounge andReynolds, J. Med. Chem., 46, (2003), 2074-2082) can be employed toestimate the attraction, repulsion, and steric hindrance of the twobinding partners (i.e. integrin α_(V)β₆ and a candidate modulator or acandidate anti-α_(V)β₆ inhibitor). Generally the tighter the fit, thefewer the steric hindrances, and the greater the attractive forces, themore potent the potential modulator since these properties areconsistent with a tighter binding constant. Furthermore, the morespecificity in the design of a potential drug, the more likely it isthat the drug will not interact with other proteins as well. This willtend to minimise potential side-effects due to unwanted interactionswith other proteins.

In one embodiment a plurality of candidate agent compounds are screenedor interrogated for interaction with the binding sites. In one example,this involves providing the structures of the candidate agent compounds,each of which is then fitted to computationally screen a database ofcompounds (such as the Cambridge Structural Database or ACD) forinteraction with the binding sites, i.e. the candidate agent compoundmay be selected by computationally screening a database of compounds forinteraction with the binding sites and containing the pharmacophoredescribed herein (see the methods in Martin, J. Med. Chem., vol 35,2145-2154 (1992)). In another example, a 3-D descriptor for the agentcompound is derived where the descriptor includes the pharmacophoricfeature(s) described herein. The descriptor may then be used tointerrogate the compound database, the identified agent compound beingthe compound, which matches with the features of the descriptor.

X-ray crystallography, NMR spectroscopy, isothermal titrationcalorimetry (ITC), thermal denaturation, mass spectrometry and surfaceplasmon resonance (SPR) assays can be used in several ways for drugdesign. The pharmacophore described herein can be used for the design,screening, development and optimization of modulators of integrinα_(V)β₆ (e.g., anti-α_(V) β₆ inhibitors).

In some embodiments, the candidate anti-α_(V)β₆ inhibitors can beidentified as binding to integrin α_(V)β₆ by using one or more of thefollowing methods.

X-ray Crystallography. Complexes of integrin α_(V)β₆ (e.g., the modifiedintegrin polypeptide dimers described herein) and a test agent can becrystallized and analyzed using X-ray diffraction methods, e.g.according to the approach described by Greer et al., J. of MedicinalChemistry, Vol. 37, (1994), 1035-1054, and difference Fourier electrondensity maps can be calculated based on X-ray diffraction patterns ofcrystals of integrin α_(V)β₆ or co-crystallized integrin α_(V)β₆ and thetest agent, as well as the solved structure of uncomplexed integrinα_(V)β₆. These maps can then be analyzed e.g. to determine whether andwhere a particular test agent binds to the integrin α_(V)β₆ and/orchanges the conformation of integrin α_(V)β₆.

Electron density maps can be calculated using programs such as thosefrom the CCP4 computing package (Collaborative Computational Project 4.The CCP4 Suite: Programs for Protein Crystallography, ActaCrystallographica, D50, (1994), 760-763.). For map visualization andmodel building programs such as “O” (Jones et al., ActaCrystallographica, A47, (1991), 110-119) or “QUANTA” (1994, San Diego,Calif.: Molecular Simulations) can be used.

The complexes can be studied using well-known X-ray diffractiontechniques and may be refined using computer software, such as CNX(Brunger et al., Current Opinion in Structural Biology, Vol. 8, Issue 5,October 1998, 606-611, and commercially available from Accelerys, SanDiego, Calif.), X-PLOR (Yale University, ©1992, distributed byAccelerys), as described by Blundell et al, (1976) and Methods inEnzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985).

This information can thus be used to optimize known classes of integrinα_(V)β₆ substrates or inhibitors, and more importantly, to design andsynthesize novel classes of anti-α_(V) β₆ selective modulators, e.g.,anti-α_(V)β₆ inhibitors.

Analysing the complex by X-ray crystallography can determine the abilityof the to set agent to interact with integrin α_(V)β₆. Analysis of theco-complexes of the integrin α_(V)β₆ may involve, e.g., phasing,molecular replacement or calculating a Fourier difference map of thecomplex as discussed above.

NMR Spectroscopy. NMR spectroscopy allows for the detection ofinteractions between ligands and a given target protein. This techniquecan be applied to detect binding, by either observing the spectrum ofthe recombinant protein, or that of the binding ligand (Lepre, Moore &Peng, Chem. Rev. 2004, 104, 3641). The former methods are currentlylimited by the size of the protein, and include the so-called ‘SAR byNMR’ method of Shuker et al (Science 274, 1531-1534 (1996)). Methodsthat observe changes in the NMR properties of the ligand are not limitedby the size of the protein, and again can be applied to the compounds.These include NMR methods that detect changes in ligand relaxation rates(T1 and T2/T1ρ) and cross-relaxation rates, either ligand-to-ligand(NOE), protein-to-ligand (trNOE and STD-NMR) or water-to-ligand(water-LOGSY) rates, on introduction of the target protein.

For example, the WaterLOGSY experiment (Dalvit et al., J. Biomol. NMR,118, 2000) can be used for finding compounds that bind with affinitiesthat are generally too weak (in the 10 M to 10 mM range) to be detectedby more conventional binding assays. The WaterLOGSY experiment is NOEbased, with a long mixing period (ca 1.5 sec) during which magnetizationcan be transferred from the protein to a binding ligand. In the absenceof protein or when a compound is not interacting with the protein, nomagnetization can be transferred from the protein to the compound, andthe rapid tumbling of the compound results in positive NOEs and negativecompound signals in the NMR spectrum. On the other hand, if a compoundis binding to the protein, magnetization is transferred from the proteinto the compound, which, due to the slow tumbling of the protein, resultsin negative NOEs and positive compound signals. Interfering proteinsignals are not observed in the experiment, due to a large excess ofcompound and the suppression of protein signals. Medium throughput canbe achieved by analysing mixtures of compounds. In order to identify theindividual compound signals in a mixture, a reference spectrum isrecorded for each compound, which can then be compared to the WaterLOGSYspectrum of the mixture. In addition, active site directed binding of acompound can be confirmed by carrying out a competition experiment witha high affinity ligand that is known to bind in the active site.Compounds binding in the active site will be displaced by the highaffinity ligand, resulting in a change from positive to negativecompound signals. The signals of compounds binding elsewhere on theprotein or non-specifically will remain positive even in the presence ofthe high affinity ligand.

A typical experiment can be conducted using a 500-1000 MHz spectrometer.For each integrin α_(V)β₆/test agent mixture a 1D reference spectrum anda WaterLOGSY spectrum is recorded. The mixtures typically contained 4-6compounds, each at a concentration of 100-300 μM. Competitionexperiments are performed by adding a high affinity compound to theintegrin α_(V) β₆/test agent mixture. The WaterLOGSY spectra of integrinα_(V) β₆/test agent mixtures in the absence and presence of the highaffinity ligand are compared. A compound can be deemed an active sitebinder only if it could be displaced by the high affinity ligand. Foreach active site binder observed in a mixture, the experiment isnormally repeated with the individual compound to confirm theobservation.

Thus, the method can further comprise the steps of: (a) obtaining orsynthesising said candidate an anti-α_(V)β₆ inhibitor; (b) forming acomplex of the integrin α_(V)β₆ and said candidate inhibitor; and (c)analysing said complex by X-ray crystallography or NMR spectroscopy todetermine the ability of said candidate inhibitor to interact with theintegrin α_(V) β₆. Surface Plasmon Resonance (SPR). Surface plasmonresonance (SPR) methods that can be used in ligand identificationinclude those methods where a protein target is immobilized on thesurface of the chip and candidate ligands passed over the protein in themobile phase (Karlsson, R., Anal. Biochem., 1994, 221, 142-151;Karlsson, R., Kullman-Magnusson, M., Hamalainen, M.-D., Remaeus, A.,Andersson, K., Borg, P., Gyzander, E., Deinum, J.; AnalyticalBiochemistry, 2000; 278, 1-13). Measurement of association anddissociation of the ligand at a range of ligand concentrations allowscalculation of ligand binding affinities (reviewed in Rich & Myszka,Curr Opin Biotechnol. 11, 54-61 (2000)).

Commercial realisations of SPR allows for the detection of interactionsbetween ligand and target protein in real time. Monitoring ofprotein/ligand interactions is done with an optical detection systembased on surface plasmon resonance (SPR). In order to measure theinteraction between ligand and protein one of the components must becovalently attached to the surface of a sensor chip. This chip iscomposed of a glass slide with a thin layer of gold deposited on oneside and a matrix such as dextran covering the gold surface. Thephenomenon of SPR occurs when light is reflected from a conducting filmthat is sandwiched between two non-absorbing media. The conducting filmis the gold layer of the chip and the two media of different refractiveindex are the glass slide and the aqueous sample flowing over thesurface of the chip. Surface plasmon resonance causes a decrease inintensity of the reflected light at a specific angle. The angle at whichthe decrease occurs is sensitive to the mass of solutes at the surfaceof the chip. When molecules bind to the surface of the chip the massincreases affecting the angle at which the decrease in intensity occurs.For example, a ligand attached to the chip surface would bind the targetprotein and increase the mass at the chip surface.

In a typical experiment, integrin α_(V)β₆/inhibitor interaction can beassayed by attaching a high affinity binder to the surface of a sensorchip. The presence of an anti-α_(V)β₆ inhibitor reduces the signal bybinding to the integrin αvβ6 and inhibiting the interaction with thehigh affinity binder on the chip. An anti-α_(V)β₆ inhibitor can thus bedetected.

Thus, the method of the invention may comprise the further steps of: (a)obtaining or synthesising said candidate anti-α_(V)β₆ inhibitor; (b)forming a complex of integrin α_(V)β₆ described herein (e.g., themodified integrin polypeptide dimers described herein) and saidcandidate inhibitor; and (c) analyzing said complex by SPR assay todetermine the ability of said candidate inhibitor to interact withintegrin α_(V)β₆.

ITC and Thermal Denaturation. Isothermal titration calorimetry (ITC) canbe used as an alternative method to detect the interaction betweenintegrin α_(V)β₆ and a candidate ligand. Again various methods have beendescribed, including direct titration of a protein solution with theligand of interest and measurement of the associated enthalpy changes(reviewed in Leavitt & Friere, Curr. Opin. Struct. Biol. 11, 560-566(2001)), low c-value methods for weak-binding ligands (Turnbull &Daranas, J Am Chem Soc. 125, 14859-66 (2003)), and competition methodsfor weak-binding ligands (Zhang & Zhang, Curr Opin Biotechnol. 11, 54-61(2000)) and extremely tight-binding ligands (Velazquez-Campoy, A., Kiso,Y., Freire, E. Arch. Biochem. Biophys. 390, 169-175 (2001).

In addition to ITC, differential scanning calorimetry (DSC) is anothercalorimetric method that can be used to identify protein ligands. ThisDSC method measures the effect of ligands upon the thermal denaturationmid-point of a target protein (Plotinov et al, Assay & Drug DevelopmentTechnologies 1, 83-89 (2002)). Thermal denaturation methods takeadvantage of the observed energetic coupling between protein stabilityand ligand binding, thus allowing identification of ligands throughtheir ability to stabilise the protein. This effect can be measured inseveral other ways, for example by using a fluorescent reporter dye tomeasure changes in the temperature at which thermal denaturation occurs(Pantoliano et al, J. Biomol. Screening 6, 429-440 (2001)) or by using afluorescent reporter dye to measure the ability of candidate ligands toalter the rate at which thermal denaturation occurs (Epps et al, Anal.Biochem. 292, 40-50 (2001)).

Thus, the method can comprise further comprise the steps of: (a)obtaining or synthesising said candidate anti-α_(V)β₆ inhibitor; (b)forming a complex of integrin α_(V)β₆ (e.g., the modified integrinpolypeptide dimers described herein) and said candidate inhibitor; and(c) analyzing said complex by thermal denaturation or ITC to determinethe ability of said candidate inhibitor to interact with integrinα_(V)β₆.

Mass Spectrometry. There are different mass spectrometry methods thathave been used to detect ligand binding to proteins. In one method, theprotein is exposed to a mixture of ligands in solution. Protein-ligandcomplexes are then separated from unbound ligands by a chromatographicmethod and the ligands identified by mass spectrometry afterdissociation of the complex (F J Moy, K Haraki, D Mobilio, G Walker, RPowers, K Tabei, H Tong, M M Siegel, Anal Chem 2001, 73, 571-581). Inanother method, the protein is exposed to single ligand or a mixture ofligands in solution. Protein-ligand complexes are detected directly byobtaining mass spectra of the complex under conditions where associationof the ligand is maintained in the mass spectrometer and the identity ofthe ligand is determined by analysis of the mass of the complex (E ESwayze, E A Jefferson, K A Sannes-Lowery, L B Blyn, L M Risen, SArakawa, S A Osgood, S A Hofstadtler and R H Griffey, J. Med. Chem.2002, 45, 3816-3819). In an alternative method, ligand binding to theprotein is detected via a change in the rate of hydrogen-deuteriumexchange when the protein is exposed to deuterate solvents in thepresence and absence of a ligand. Various experimental schemes arepossible which measure global or local changes in exchange caused by thepresence of a ligand (K D Powell, S Ghaemmaghami, M Z Wang, L Ma, T GOas and M C Fitzgerald JACS 2002, 124, 10256-10257; M M Zhu, D L Rempel,Z Du, M L Gross, JACS 2003, 125, 5253-5253).

By introducing at least one disulfide bond to an integrin dimer, theinteraction between the integrin α subunit and the integrin β subunit,unlike the wild-type integrin dimer, is irreversible, and thus crystalstructure of the disulfide-linked integrin dimer can be formed with ahigh resolution, which can then used for various applications, e.g.,pharmacophore modeling, and/or drug screening. Accordingly, in anotheraspect, provided herein is a method of identifying an anti-α_(V)β₆inhibitor. The method comprises: (a) providing or generating, on acomputer, a three-dimensional structure of α_(V)β₆ integrin protein or aportion thereof characterized by atomic structure coordinates (e.g., asdescribed in Table 6 of, Application No. 62/033,699 or athree-dimensional structure that exhibits a root-mean-square difference(rmsd) in α-carbon positions of less than 2.0 Å (or less than 1.0 Å)with the atomic structure coordinates (e.g., as described in Table 6 ofPCT Application No: PCT/US15/44093 filed Aug. 6, 2015; (b) docking onthe computer a first molecular entity in a first part of a bindingpocket of the integrin β₆ headpiece polypeptide having an amino acidsequence of SEQ ID NO: 2, to determine the binding association betweenthe first molecular entity and the first part of the binding pocket,wherein the binding pocket comprises amino acid residues Ala-217,Asn-218, Pro-179, Cys-180, Ile-183, Ala-126, and Tyr-185; (c) docking onthe computer a second molecular entity in a second part of the bindingpocket, to determine the binding association between the secondmolecular entity and the second part of the binding pocket; (d)repeating steps (b) to (c) with a different first and second molecularentity; (e) selecting a first and a second chemical entity based on thequantified binding associations; and (f) generating on the computer apharmacophore model by assembling the selected first and secondmolecular entity into a molecular representation that interacts with thebinding pocket.

In another aspect, a method of identifying an anti-α_(V)β₃ inhibitor isalso provided herein. The method comprises: (a) providing, on acomputer, a three-dimensional crystalline structure of α_(V)β₃ integrinprotein or a portion thereof characterized by its atomic structurecoordinates(e.g., as described in Table 8 of PCT Application No:PCT/US15/44093 filed Aug. 6, 2015), or a three-dimensional structurethat exhibits a root-mean-square difference (rmsd) in α-carbon positionsof less than 2.5 Å (or less than 2.0 Å, or less than 1.0 Å) with itsatomic structure coordinates (e.g., as described in Table 8 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015); (b) docking on thecomputer a first molecular entity in a first part of a binding pocket ofthe integrin β₃ headpiece polypeptide having an amino acid sequence ofSEQ ID NO: 5 or a fragment thereof (with desired domain(s)), todetermine the binding association between the first molecular entity andthe first part of the binding pocket; (c) docking on the computer asecond molecular entity in a second part of the binding pocket, todetermine the binding association between the second molecular entityand the second part of the binding pocket; (d) repeating steps (b) to(c) with a different first and second molecular entity; (e) selecting afirst and a second chemical entity based on the quantified bindingassociations; and (f) generating on the computer a pharmacophore modelby assembling the selected first and second molecular entity into amolecular representation that interacts with the binding pocket.

As used herein, the term “molecular entity” refers to an atom, amolecule, or a chemical functional group. Various molecular entities areknown in the art, including, e.g., but not limited to, hydrophobicmoieties, acidic moieties, basic moieties, polar moieties, non-polarmoieties, negatively-charged moieties, positively-charged moieties, andany combinations thereof, and can be used in the docking analysis todetermine a pharmacophore model. Non-limiting examples of molecularentities include hydrogen, alkyl, aryl, halogen, hydroxyl, amino,alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, alkenyl,alkynyl, keto, carbonyl, amide, amidine, guanidine, glutamyl, nitrate,nitro, nitrile, azido, sulfide, disulfide, sulfone, sulfoxide, and anycombinations thereof.

As used herein, the term “docking” refers to manipulation or placementof a molecular entity in a binding pocket. For example, docking caninclude orienting, rotating, translating a molecular entity in a bindingpocket, domain, molecule or molecular complex or portion thereof basedon distance geometry and/or energy. In some embodiments, docking can beperformed by distance geometry methods that find sets of atoms of amolecular entity that match sets of sphere centers of the bindingpocket, domain, molecule or molecular complex or portion thereof. SeeMeng et al. J. Comp. Chem. 4: 505-524 (1992). Sphere centers aregenerated by providing an extra radius of given length from the atoms(excluding hydrogen atoms) in the binding pocket, domain, molecule ormolecular complex or portion thereof. Real-time interaction energycalculations, energy minimizations or rigid-body minimizations (Gschwendet al., J. Mol. Recognition 9:175-186 (1996)) can be performed whileorienting the chemical entity to facilitate docking. For example,interactive docking experiments can be designed to follow the path ofleast resistance. If the user in an interactive docking experiment makesa move to increase the energy, the system will resist that move.However, if that user makes a move to decrease energy, the system willfavor that move by increased responsiveness. (Cohen et al., J. Med.Chem. 33:889-894 (1990)). Docking can also be performed by combining aMonte Carlo search technique with rapid energy evaluation usingmolecular affinity potentials. See Goodsell and Olson, Proteins:Structure, Function and Genetics 8:195-202 (1990). Software programsthat carry out docking functions include but are not limited to MATCHMOL(Cory et al., J. Mol. Graphics 2: 39 (1984); MOLFIT (Redington, Comput.Chem. 16 217 (1992)) and DOCK (Meng et al., supra).

The term “providing or generating a three-dimensional structure” as usedherein refers to converting a list of atomic structure coordinates intoa structural model or graphical representation in a three-dimensionalspace. This can be achieved, for example, through commercially orpublicly available software. A model of a three-dimensional structure ofa molecule or molecular complex can thus be constructed on a computerthat contains the atom structure coordinates of a desired molecule ormolecular complex and appropriate software. The three-dimensionalstructure can be displayed or used to perform computer modeling orfitting or docking operations. In some embodiments, the atomic structurecoordinates themselves, without the displayed model, can be used toperform computer-based modeling and/or fitting or docking operations.

As used herein, the term “binding association” refers to a measure ofshape complementarity between a molecular entity and at least part of abinding pocket, which is correlated with a superimposition between allor part of the atoms of a molecular entity and/or all or part of theatoms of a ligand bound in a binding pocket of a molecule of interest.In some embodiments, the docking process can be facilitated by RMSDvalues and/or calculations of distance geometry and/or energy. Energycan include but is not limited to interaction energy, free energy anddeformation energy. See Cohen, supra. For example, if a molecular entitymoves to an orientation with a high RMSD, the system will resist themotion.

The term “root mean square deviation” or “RMSD” means the square root ofthe arithmetic mean of the squares of the deviations from the mean. Itis a way to express the deviation or variation from a trend or object.For purposes of pharmacophore modeling, the “root mean square deviation”defines the variation in the backbone atoms of an integrin heterodimer,e.g., for integrin α_(V) β₆, a binding pocket, a headpiece a motif, adomain, or portion thereof, as defined by the atomic structurecoordinates of a crystal structure of integrin α_(V)β₆ (e.g., describedin Table 6 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015, orfor integrin α_(V)β₃, a binding pocket, a headpiece a motif, a domain,or portion thereof, as defined by the atomic structure coordinates of acrystal structure of integrin α_(V)β₃ (e.g., as described in Table 8 ofPCT Application No: PCT/US15/44093 filed Aug. 6, 2015). It would beapparent to a skilled artisan that the calculation of RMSD involves astandard error of 0.1 Å.

After determining a possible candidate anti-α_(V)β₆ inhibitor oranti-α_(V)β₃ inhibitor from a pharmacophore model, the method of thisaspect and other aspects described herein can further comprise the stepsof: (a) obtaining or synthesising said candidate anti-α_(V)β₆ inhibitoror anti-α_(V)β₃ inhibitor; (b) forming a complex of integrin α_(V)β₆ orα_(V)β₃ and said candidate inhibitor; and (c) analyzing said complex bymass spectrometry to determine the ability of said candidate modulatorto interact with integrin α_(V)β₆ or α_(V)β₃.

In some embodiments, the method can further comprise contacting thecandidate anti-integrin inhibitor (e.g., anti-α_(V)β₆ inhibitor oranti-α_(V)β₃ inhibitor) with an integrin protein (e.g., an α_(V)β₆integrin protein or an α_(V)β₃ integrin protein) to determine theability of the candidate anti-integrin inhibitor (e.g., anti-α_(V)β₆integrin inhibitor or anti-α_(V)β₃ inhibitor) to bind the correspondingintegrin protein (e.g., α_(V)β₆ integrin protein or α_(V)β₃ integrinprotein).

In some embodiments, biological assays or cells assays can be performedto determine the ability of the anti-integrin inhibitor (e.g.,anti-α_(V)β₆ integrin inhibitor or anti-α_(V)β₃ integrin inhibitor) tobind the corresponding integrin protein (e.g., (α_(V)β₆ integrin proteinor α_(V)β₃ integrin protein). For example, the method can furthercomprise contacting cells of interest with the candidate anti-integrininhibitor (e.g., anti-α_(V)β₆ inhibitor or anti-α_(V)β₃ inhibitor) anddetecting the response of the cells, e.g., biological propertiesassociated with integrin signaling (e.g., α_(V)β₆ or α_(V)β₃ integrinsignaling), e.g., but not limited to cell adhesion, migration potential,cell viability, production of cytokines, cell morphology, and anycombinations thereof.

Following identification of anti-integrin inhibitors (e.g., anti-α_(V)β₆inhibitors or anti-α_(V)β₃ inhibitors) using the pharmacophore modeldescribed herein, the inhibitors can be manufactured and/or used in thepreparation, i.e. manufacture or formulation, of a composition such as amedicament, pharmaceutical composition or drug. These can beadministered to individuals who are in need thereof.

The methods described herein to identify a ligand for an integrinα_(V)β₆ heterodimer or an anti-α_(V)β₆ inhibitor can be extended toidentify a ligand or an inhibitor for other integrin αβ heterodimers,e.g., α_(V)β₃ or α_(V)β₈ heterodimers. For example, a pharmacophore of aligand or an inhibitor for other integrin αβ heterodimer can be designedon the identification of a binding pocket of the crystal structure ofthe corresponding modified integrin polypeptide dimer described herein.

Crystalline Compositions

In a further aspect, provided herein is a crystalline compositioncomprising a ligand-binding headpiece of integrin α_(V)β₆, wherein thecrystalline composition is characterized with space group C2, and hasunit cell parameters a=184.5±3 Å, b=168.3±3 Å, c=101.8±3 Å, α=β=90°, andγ=98.2°±3°.

In some embodiments, the crystalline composition can further comprise aligand. Accordingly, a crystalline composition comprising aligand-binding headpiece of integrin α_(V)β₆ and a ligand is alsoprovided herein. The crystalline composition is characterized with spacegroup C2, and has unit cell parameters a=184.4±3 Å, b=170.0±3 Å,c=102.4±3 Å, α=β=90°, and γ=98.7° °±3°.

In some embodiments, the ligand-binding headpiece of integrin α_(V)β₆can comprise a modified integrin α_(V) headpiece polypeptide describedherein, and a modified integrin β₆ headpiece polypeptide describedherein.

In some embodiments, the crystalline composition has a binding pocket inthe modified integrin β₆ headpiece polypeptide, wherein the bindingpocket comprises amino acid residues Ala-217, Asn-218, Pro-179, Cys-180,Ile-183, Ala-126, and Tyr-185.

In some embodiments, the ligand-binding headpiece of integrin β₆ can bedescribed by its atomic structure coordinates (e.g., described in Table6 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015), or astructure that exhibits a root-mean-square difference (rmsd) in α-carbonpositions of less than 2.0 Å (or less than 1.0 Å) with its atomicstructure coordinates

In some embodiments where the crystalline composition further comprisesa ligand, e.g., a peptide, the crystalline composition can be describedby its atomic structure coordinates (e.g., described in Table 7 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015), or a structure thatexhibits a root-mean-square difference (rmsd) in α-carbon positions ofless than 2.0 Å (or less than 1.0 Å) with its atomic structurecoordinates.

A crystalline composition comprising a ligand-binding headpiece ofintegrin α_(V)β₃ is also provided herein. The crystalline composition ischaracterized with space group P22121, and has unit cell parametersa=87±2 Å, b=124±2 Å, c=165±2 Å, α=β=90°, and γ=90°±3°. In someembodiments, the ligand-binding headpiece of integrin α_(V)β₃ cancomprise a modified integrin α_(V) headpiece polypeptide describedherein, and a modified integrin β₃ headpiece polypeptide describedherein. In some embodiments, the ligand-binding headpiece of integrinα_(V)β₃ can be described by its atomic structure coordinates (e.g., asdescribed in Table 8 of PCT Application No: PCT/US15/44093 filed Aug. 6,2015), or a structure that exhibits a root-mean-square difference (rmsd)in α-carbon positions of less than 2.5 Å (or less than 2.0 Å, or lessthan 1.0 Å) with its atomic structure coordinates (e.g., as described inTable 8 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015).

Another aspect provided herein relates to a crystalline compositioncomprising a ligand-binding headpiece of integrin α_(V)β₈. Theligand-binding headpiece of integrin α_(V)β₈ is characterized with spacegroup β₁, and has unit cell parameters a=153±3 Å, b=55±3 Å, c=181±3 Å,α=β=90°, and γ=110°±3°. In some embodiments, the ligand-bindingheadpiece of integrin α_(V)β₈ can comprise a modified integrin α_(V)headpiece polypeptide described herein, and a modified integrin β₈headpiece polypeptide described herein.

In some embodiments of various aspects described herein, the crystallinecomposition can be formed from a crystallization solution bufferedbetween pH 6-8 at a temperature of about 20° C. or at room temperature,and having an ionic strength of about 800-900 mM.

As used herein, the term “crystallization solution” refers to a solutionwhich promotes crystallization comprising at least one agent, e.g., butnot limited to a buffer, one or more salts, a precipitating agent, oneor more detergents, sugars or organic compounds, lanthanide ions, apoly-ionic compound, a stabilizer, or any combinations thereof.

In some embodiments, the crystallization solution for forming integrinα_(V)β₆ headpiece can comprise 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mMCaCl₂ and 1 mM MgCl₂ buffer (1 μl) and 1 μl reservoir solution of 20%PEG 4000, 0.1 M sodium cacodylate pH 6.0, 0.2 M ammonium sulfate.

The crystalline compositions can be used for various applications,including, e.g., drug screening or pharmacophore modeling. Thus, forexample, in one aspect, a method of identifying an anti-α_(V)β₈inhibitor is also provided herein. The method comprises: (a) providing,on a computer, a three-dimensional crystalline structure of α_(V)β₈integrin protein or a portion thereof derived from the crystallinecomposition comprising a ligand-binding headpiece of integrin α_(V)β₈described herein; (b) docking on the computer a first molecular entityin a first part of a binding pocket of the integrin β₈ headpiecepolypeptide having an amino acid sequence of SEQ ID NO: 6 or a fragmentthereof (e.g., with desired domain(s)), to determine the bindingassociation between the first molecular entity and the first part of thebinding pocket; (c) docking on the computer a second molecular entity ina second part of the binding pocket, to determine the bindingassociation between the second molecular entity and the second part ofthe binding pocket; (d) repeating steps (b) to (c) with a differentfirst and second molecular entity; (e) selecting a first and a secondchemical entity based on the quantified binding associations; and (f)generating on the computer a pharmacophore model by assembling theselected first and second molecular entity into a molecularrepresentation that interacts with the binding pocket. In someembodiments, the method can further comprise contacting the candidateanti-α_(V)β₈ inhibitor (based on the pharmacophore model) with anα_(V)β₈ integrin protein to determine the ability of the candidateanti-α_(V)β₈ integrin inhibitor to bind the α_(V)β₈ integrin protein.While the method in this aspect is directed to identifying ananti-α_(V)β₈ inhibitor, the same method can be used to identify otheranti-integrin inhibitor (e.g., anti-α_(V)β₃ inhibitor or anti-α_(V)β₆inhibitor) with the appropriate crystalline compositions describedherein.

Applicants have provided the crystal structure coordinates for integrinα_(V)β₆ in Table 6 of PCT Application No: PCT/US15/44093 filed Aug. 6,2015 including, (a) “ATOM” records, which lists the amino acid residuesin integrin α_(V)β₆ headpiece and their atomic structure coordinates ina three-dimensional space characterized by x, y, and z coordinates; (b)“ANISOU” records, which present the anisotropioc temperature factorsrelating to the corresponding ATOM isotropic temperature factors asb-factor (e.g., in column 11 of “ATOM” records in Table 6 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015); and (c) “HETA™”records, which provide the atomic coordinate records for atoms within“non-standard” groups, e.g., water molecules and atoms presented inheterogen groups such as prosthetic groups, inhibitors, solventmolecules, and ions. SEQ ID NOS 115-121 are disclosed herein and Table 6of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015, respectively,in order of appearance.

Applicants have provided the crystal structure coordinates for integrinα_(V)β₆ headpiece and a peptide in Table 7 of PCT Application No:PCT/US15/44093 filed Aug. 6, 2015 including, (a) “ATOM” records, whichlists the amino acid residues in a complex comprising an integrinα_(V)β₆ headpiece and a peptide, and their atomic structure coordinatesin a three-dimensional space characterized by x, y, and z coordinates;(b) “ANISOU” records, which present the anisotropioc temperature factorsrelating to the corresponding ATOM isotropic temperature factors asb-factor (e.g., in column 11 of “ATOM” records in Table 7 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015; and (c) “HETA™”records, which provide the atomic coordinate records for atoms within“non-standard” groups, e.g., water molecules and atoms presented inheterogen groups such as prosthetic groups, inhibitors, solventmolecules, and ions. The anisotropic temperature factors are stored inthe same coordinate frame as the atomic coordinate records. SEQ ID NOS115-120, 122-128, 121, and 78, are disclosed herein and Table 7 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015 respectively, in orderof appearance.

Applicants have provided the crytal structure coordinates for α_(V)β₃ inTable 8 of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015,including, (a) “ATOM” records, which lists the amino acid residues in anintegrin α_(V)β₃ headpiece, and their atomic structure coordinates in athree-dimensional space characterized by x, y, and z coordinates; (b)“ANISOU” records, which present the anisotropioc temperature factorsrelating to the corresponding ATOM isotropic temperature factors asb-factor in column 11 of “ATOM” records in (e.g., Table 8 of PCTApplication No: PCT/US15/44093 filed Aug. 6, 2015); and (c) “HETA™”records, which provide the atomic coordinate records for atoms within“non-standard” groups, e.g., water molecules and atoms presented inheterogen groups such as prosthetic groups, inhibitors, solventmolecules, and ions. The anisotropic temperature factors are stored inthe same coordinate frame as the atomic coordinate records. Additionalinformation about the coordinate record description (e.g., Tables 6-7 ofPCT Application No: PCT/US15/44093 filed Aug. 6, 2015 in PDB format) canbe accessed online at RCSB protein data bank website, which isincorporated herein by reference in their entirities. SEQ ID NOS 129-130are disclosed herein and Table 8 of PCT Application No: PCT/US15/44093filed Aug. 6, 2015, respectively, in order of appearance.

Different protein chains are specified by the “TER” keyword, as well asa one-letter designation in the coordinate records (see column 22: ChainID). The chains are included one after another separated by a TER recordto indicate that the chains are not physically connected to each other,i.e., no covalent bond connecting different chains.

The b-factor or temperature factor is a value to account fordistribution of electrons in a non-ideal situation, e.g., due tovibration of atoms or differences between the many different moleculesin a crystal lattice, which yields a slightly smeared image of themolecule. The amount of smearing is proportional to the magnitude of theb-factor. Thus, b-factor can be a measure of confidence in the locationof each atom or molecule. Values under 10 generally create a model ofthe atom that is very sharp, indicating that the atom is not moving muchand is in the same position in all of the molecules in the crystal.Values greater than 50 or so generally indicate that the atom is movingso much that it can barely been seen. For example, in the case withatoms at the surface of proteins, long sidechains are free to wag in thesurrounding. These atoms generally have high b-factor, and theircoordinates e.g., as specified in Table 6 of PCT Application No:PCT/US15/44093 filed Aug. 6, 2015, may represent only one possiblesnapshot of its location.

Macromolecular crystals are composed of many individual molecules packedinto a symmetrical arrangement. The term “occupancy” as used in “ATOM”records provides an estimation of the number of conformations of eachamino acid residue observed in a crystal lattice. An occupancy value ofabout 1 indicates that the amino acid residue is found at the same placeof all of the molecules packed into the crystal.

Kits for Performing Various Aspects of the Methods Described Herein

Kits comprising materials for performing methods according to variousaspects described herein are also provided herein. A kit can be in anyconfiguration well known to those of ordinary skill in the art and canbe used to perform one or more of the methods described herein, e.g.,for forming a modified integrin polypeptide dimer (e.g., for identifyinga binding site or pocket for a ligand), determining whether a test agentforms a complex with an integrin heterodimer, and/or determining bindingaffinity of a test agent to an integrin heterodimer.

In one aspect, the kit comprises, consists essentially of, or consistsof either one or both of the modified integrin α and β headpiecepolypeptides or functional fragments/variants described herein (e.g.,but not limited to the modified integrin α_(V) headpiece polypeptidesdescribed herein, and/or the modified integrin β₆ headpiece polypeptidesdescribed herein), and at least one reagent and/or purification device(e.g., one or more standard protein purification columns). In someembodiments, the kit can comprise, consist essentially of, or consist ofat least one of the modified integrin α headpiece polypeptides describedherein or a functional fragment thereof, at least one of the modifiedintegrin β headpiece polypeptides described herein or a functionalfragment thereof, and at least one reagent. In some embodiments, the kitcan comprise, consist essentially of, or consist of at least two or moredifferent modified integrin α headpiece polypeptides described herein orfunctional fragments thereof, at least two or more modified integrin βheadpiece polypeptides described herein or a functional fragmentsthereof, and at least one reagent. By providing different modifiedintegrin α and/or β headpiece polypeptides or functional fragmentsthereof, different modified integrin polypeptide dimers can be producedaccording to a user's need.

In some embodiments, the reagent included in the kit can be anymaterials or solutions to form a modified integrin polypeptide dimertherein. For example, the reagent can comprise a buffered solution,e.g., suitable for forming a modified integrin polypeptide dimertherein.

In some embodiments, the kit can further comprise packaging materialsand instructions for forming a modified integrin polypeptide dimer fromthe modified integrin α and/or β headpiece polypeptides provided in thekit.

In another aspect, the kit comprises, consists essentially of, orconsists of at least one or more modified integrin polypeptide dimersdescribed herein, e.g., the modified integrin α_(V)β₆ dimer describedherein, and and at least one reagent. In some embodiments, the kit canbe used to identify a binding site or pocket for a ligand or aninhibitor. In some embodiments, the kit can be used to determine whethera test agent of a user's choice forms a complex with an integrinheterodimer. In some embodiments, the kit can be used to determinebinding affinity of a test agent of a user's choice to an integrinheterodimer. In some embodiments, the kit can be used to identify aligand or an inhibitor for the modified integrin polypeptide dimersdescribed herein.

In some embodiments, the kit can comprise at least two or more differentmodified integrin polypeptide dimers described herein, including, butnot limited to, the modified integrin α_(V)β₆ dimer described herein,and the modified integrin α_(V)β₃ dimer described herein, and and atleast one reagent. In these embodiments, the kit can also be used tocharacterize effects of a test agent on more than one integrinheterodimers. For example, the kit can be used to determine thespecificity of a test agent to interact with an integrin heterodimer.

In some embodiments, the reagent included in the kit of this aspect canbe any materials or solutions suitable to maintain the biologicalactivity of the modified integrin polypeptide dimers included in the kitand/or to perform downstream applications as noted above. For example,the reagent can comprise a buffered solution, e.g., suitable forperforming crystallization of a modified integrin headpiece polypeptidedescribed herein (complexed with or without a test agent) and detectingthe crystal structure by X-ray crystallography, fluorescence anisotropy,and/or flow cytometry. Reagents and/or materials commonly used indetecting a protein crystal structure by X-ray crystallography,fluorescence anisotropy, and/or flow cytometry are known in the art andcan be included in the kit described herein.

In some embodiments, the kit can further comprise packaging materialsand instructions for performing crystallization of a modified integrinpolypeptide dimer described herein (complexed with or without a testagent) and detecting the crystal structure by X-ray crystallography. Insome embodiments, the kit can further comprise packaging materials andinstructions for determining integrin heterodimer-test agent interaction(including, e.g., formation of an integrin heterodimer-test agentcomplex, and/or measurement of a binding affinity of a test agent to amodified integrin polypeptide dimer described herein), for example, byperforming fluorescence anisotropy, and/or flow cytometry.

In some embodiments, the kit can further comprise at least one or morecompeting agent described herein.

In various aspects of the kits described herein, the modified integrin αor β headpiece polypeptides described herein, and/or the modifiedintegrin polypeptide dimers described herein can be present inparticles, lyophilized powder, solution, or suspension.

In some embodiments of various aspects described herein, the modifiedintegrin headpiece polypeptides and/or the modified integrin polypeptidedimers included in the kit can be attached to a solid surface. Dependingon the need of desired applications, the solid surface can be made ofany material, including, but are not limited to, glass, silicone,cellulose-based materials (e.g., paper), plastics, polymer, and/or anycombinations thereof. In some embodiments, the kit can further comprisea microtiter plate, wherein each well of the microtiter plate is coatedwith at least one or more of the modified integrin polypeptides and/orthe modified integrin polypeptide dimers described herein.

In all such embodiments of various aspects described herein, the kitincludes the necessary packaging materials and informational materialtherein to use said kits. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of a compound(s) described herein forthe methods described herein. In one embodiment, the informationalmaterial can include information about production and/or molecularweight of the modified integrin headpiece polypeptides or modifiedintegrin polypeptide dimers described herein, concentration, date ofexpiration, batch or production site information, and so forth. In oneembodiment, the informational material relates to methods for forming amodified integrin polypeptide dimer and/or a crystal structure thereof.

In one embodiment, the informational material can include instructionsto form a modified integrin polypeptide dimer and/or a crystal structurethereof in a suitable manner to perform the methods described herein,e.g., for identifying a binding site or pocket for a ligand, determiningwhether a test agent forms a complex with an integrin heterodimer,and/or determining binding affinity of a test agent to an integrinheterodimer.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In all embodiments of the aspects described herein, the kit willtypically be provided with its various elements included in one package,e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a styrofoambox. The enclosure can be configured so as to maintain a temperaturedifferential between the interior and the exterior, e.g., it can provideinsulating properties to keep the reagents at a preselected temperaturefor a preselected time. The kit can include one or more containers forthe composition containing a compound(s) described herein. In someembodiments, the kit contains separate containers (e.g., two separatecontainers for the two agents), dividers or compartments for thecomposition(s) and informational material. For example, the compositioncan be contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of acompound described herein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of a compound described herein. The containers of thekits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

Exemplary Methods for Producing the Modified Integrin α HeadpiecePolypeptides, Modified Integrin β Headpiece Polypeptides, ModifiedIntegrin Polypeptide Dimers Described Herein

The modified integrin α headpiece polypeptides, modified integrin βheadpiece polypeptides, and/or modified integrin polypeptide dimersdescribed herein can be produced by any protein modifications and/or DNArecombinant methods known in the art. For example, by engineering a cellto comprise a nucleic acid, e.g., an isolated nucleic acid, encoding amodified integrin α headpiece polypeptide described herein and/or amodified integrin β headpiece polypeptide described herein; culturingthe cell under conditions suitable for the production of the modifiedpolypeptides; and isolating and/or purifying the modified polypeptides,e.g., by affinity purification, various modified integrin α headpiecepolypeptides, modified integrin β headpiece polypeptides, and/ormodified integrin polypeptide dimers described herein can be produced,e.g., for use in the methods described herein, and/or for inclusion inthe kits described herein.

Nucleic acid molecules encoding a modified integrin α headpiecepolypeptide described herein and/or a modified integrin β headpiecepolypeptide described herein can be prepared by a variety of methodsknown in the art. These methods include, but are not limited to, PCT,ligation, and direct synthesis. A nucleic acid sequence encoding apolypeptide as described herein can be recombined with vector DNA inaccordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, e.g., by Maniatis et al., Molecular Cloning, Lab. Manual(Cold Spring Harbor Lab. Press, N Y, 1982 and 1989), and Ausubel, 1987,1993, and can be used to construct nucleic acid sequences which encode aheme-binding molecule and/or composition polypeptide as describedherein.

The term “vector” encompasses any genetic element that is capable ofreplication when associated with the proper control elements and thatcan transfer gene sequences to cells. A vector can include, but is notlimited to, a cloning vector, an expression vector, a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

In one aspect, provided herein is an expression vector comprising anucleic acid encoding a modified integrin α headpiece polypeptidedescribed herein and/or a modified integrin β headpiece polypeptidedescribed herein. Such vectors can be ued, e.g. to transform a cell inorder to produce the encoded polypeptide. As used herein, the term“expression vector” refers to a vector that directs expression of an RNAor polypeptide from sequences linked to transcriptional regulatorysequences on the vector. The sequences expressed will often, but notnecessarily, be heterologous to the cell. An expression vector maycomprise additional elements, for example, the expression vector mayhave two replication systems, thus allowing it to be maintained in twoorganisms, for example in mammalian cells for expression and in aprokaryotic host for cloning and amplification. The term “expression”refers to the cellular processes involved in producing RNA and proteinsand as appropriate, secreting proteins, including where applicable, butnot limited to, for example, transcription, transcript processing,translation and protein folding, modification and processing.“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene.The term “gene” means the nucleic acid sequence, which is transcribed(DNA) to RNA in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

Examples of vectors useful in delivery of nucleic acids encodingisolated peptides as described herein include plasmid vectors, non-viralplasmid vectors (e.g. see U.S. Pat. Nos. 6,413,942, 6,214,804,5,580,859, 5,589,466, 5,763,270 and 5,693,622, all of which areincorporated herein by reference in their entireties); retroviruses(e.g. see U.S. Pat. No. 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-52; Miller et al., Meth. Enzymol.217:581-599 (1993); Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop.3:102-09. Boesen et al., Biotherapy 6:291-302 (1994); Clowes et al., J.Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossmanand Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993), thecontents of each of which are herein incorporated by reference in theirentireties); lentiviruses (e.g., see U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, the contents of which are herein incorporatedby reference in their entireties; adenovirus-based expression vectors(e.g., see Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al.(1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994)Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; andRich et al. (1993) Human Gene Therapy 4:461-76; Wu et al. (2001)Anesthes. 94:1119-32; Parks (2000) Clin. Genet. 58:1-11; Tsai et al.(2000) Curr. Opin. Mol. Ther. 2:515-23; and U.S. Pat. Nos. 6,048,551;6,306,652 and 6,306,652, incorporated herein by reference in theirentireties); Adeno-associated viruses (AAV) (e.g. see U.S. Pat. Nos.5,139,941; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contentsof each of which are incorporated by reference herein in theirentireties); and avipox vectors (e.g. see WO 91/12882; WO 89/03429; andWO 92/03545; which are incorporated by reference herein in theirentireties).

Useful methods of transfection can include, but are not limited toelectroporation, sonoporation, protoplast fusion, peptoid delivery, ormicroinjection. See, e.g., Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratories, New York, for adiscussion of techniques for transforming cells of interest; andFelgner, P. L. (1990) Advanced Drug Delivery Reviews 5:163-87, for areview of delivery systems useful for gene transfer. Exemplary methodsof delivering DNA using electroporation are described in U.S. Pat. Nos.6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No.2002/0146831, and International Publication No. WO/0045823, all of whichare incorporated herein by reference in their entireties.

Non-limiting examples of vectors useful for expression in prokaryoticcells can include plasmids. Plasmid vectors can include, but are notlimited to, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18,pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− orKS+/−(see “Stratagene Cloning Systems” Catalog (1993) from Stratagene,La Jolla, Calif., which is hereby incorporated by reference), pQE,pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymeraseto Direct Expression of Cloned Genes,” Gene Expression Technology, vol.185 (1990), which is hereby incorporated by reference in its entirety).Non-limiting examples of mammalian and insect appropriate vectors caninclude pcDNA3, pCMV6, pOptiVec, pFUSE, and pFastBac.

The cell comprising the nucleic acid can be, e.g. a microbial cell or amammalian cell. In some embodiments, the cell as described herein iscultured under conditions suitable for the expression of the modifiedintegrin α headpiece polypeptides described herein, the modifiedintegrin β headpiece polypeptides described herein, and/or the modifiedintegrin polypeptide dimers described herein. Such conditions caninclude, but are not limited to, conditions under which the cell iscapable of growth and/or polypeptide synthesis. Conditions may varydepending upon the species and strain of cell selected. Conditions forthe culture of cells, e.g. prokaryotic and mammalian cells, are wellknown in the art. In some embodiments, the cell for expressing amodified integrin α headpiece polypeptide described herein, a modifiedintegrin β headpiece polypeptide described herein, and/or a modifiedintegrin polypeptide dimer can be HEK 293 GnTI⁻ cells.

Some Selected Definitions

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” with respect to numerical values means within5%

The term “wild type” refers to the naturally-occurring polypeptidesequence as it normally exists in vivo.

The term “polypeptide” refers to an isolated polymer of amino acidresidues, and are not limited to a minimum length unless otherwisedefined. Peptides, oligopeptides, dimers, multimers, and the like, arealso composed of linearly arranged amino acids linked by peptide bonds,and whether produced biologically and isolated from the naturalenvironment, produced using recombinant technology, or producedsynthetically typically using naturally occurring amino acids.

As used herein, the term “modified,” when referring to a polypeptide orprotein, e.g., the modified integrin α headpiece polypeptides describedherein, the modified integrin β headpiece polypeptides described herein,and/or the modified integrin polypeptide dimers described herein, meansthe polypeptide or protein including at least one or more modifications,such as deletions, additions, and substitutions (generally conservativein nature as would be known to a person in the art), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications can include, e.g., site-directed mutagenesis, and/ormutations of hosts that produce the proteins during recombinant DNAmethods.

As used interchangeably herein and throughout the specification, theterms “heterodimer” and “dimer” refer to a polypeptide or proteincomprising two or more different subunits. In the context of variousaspects described herein, the term “heterodimer” or “dimer” refers to apolypeptide or protein structure comprising, consisting esstentially of,or consisting of a wild-type or modified integrin α headpiecepolypeptide and a wild-type or modified integrin β headpiecepolypeptide. With respect to naturally occurring integrin heterodimer ordimer, a wild-type integrin α headpiece polypeptide and a wild-typeintegrin β headpiece polypeptide are non-covalently linked to eachother. With respect to modified integrin polypeptide dimers describedherein, in some embodiments, a modified integrin α headpiece polypeptideand a wild-type integrin β headpiece polypeptide are covalently linkedto each other via at least one disulfide bond. In some embodiments, amodified integrin polypeptide dimer can comprise, consist essentiallyof, or consist of a wild-type integrin α headpiece polypeptide and amodified integrin β headpiece polypeptide covalently linked together viaat least one disulfide bond. In some embodiments, a modified integrinpolypeptide dimer can comprise, consist essentially of, or consist of amodified integrin α headpiece polypeptide and a modified integrin βheadpiece polypeptide covalently linked together via at least onedisulfide bond.

The term “substitution” when referring to an amino acid sequence, refersto a change in an amino acid for a different entity, for example anotheramino acid or amino-acid moiety. Substitutions can be conservative ornon-conservative substitutions.

As used herein, the term “test agent” generally refers to a molecule,compound, or composition to be screened in one or more methods describedherein (e.g., for binding capability and/or affinity with one or moremodified integrin polypeptide dimers described herein). The test agentcan be a protein, a peptide, an antibody, a nucleic acid molecule, anaptamer, a peptidomimetic, a small molecule, or any combinationsthereof.

The term “antibody” as used herein refers to a full length antibody orimmunoglobulin, IgG, IgM, IgA, IgD or IgE molecules, or a proteinportion thereof that comprises only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind a target, such as an epitope orantigen. Examples of portions of antibodies or epitope-binding proteinsencompassed by the present definition include: (i) the Fab fragment,having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is aFab fragment having one or more cysteine residues at the C-terminus ofthe CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv)the Fd′ fragment having VH and CH1 domains and one or more cysteineresidues at the C terminus of the CH1 domain; (v) the Fv fragment havingthe VL and VH domains of a single arm of an antibody; (vi) the dAbfragment (Ward et al., 341 Nature 544 (1989)) which consists of a VHdomain or a VL domain that binds antigen; (vii) isolated CDR regions orisolated CDR regions presented in a functional framework; (viii) F(ab′)₂fragments which are bivalent fragments including two Fab′ fragmentslinked by a disulphide bridge at the hinge region; (ix) single chainantibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242Science 423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161;Hollinger et al., 90 PNAS 6444 (1993)); (xi) “linear antibodies”comprising a pair of tandem Fd segments (VH-CH1-VH—CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al., 8 Protein Eng. 1057 (1995); and U.S.Pat. No. 5,641,870).

“Antibodies” include antigen-binding portions of antibodies such asepitope- or antigen-binding peptides, paratopes, functional CDRs;recombinant antibodies; chimeric antibodies; tribodies; midibodies; orantigen-binding derivatives, analogs, variants, portions, or fragmentsthereof.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a target molecule, orto a molecule in a signaling pathway that modulates the expressionand/or activity of a target molecule. The generation and therapeutic useof aptamers are well established in the art. See, e.g., U.S. Pat. No.5,475,096.

As used here in, the term “peptidomimetic” means a peptide-like moleculethat has the activity of the peptide on which it is structurally based.Such peptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,and have an activity such as the cardiac specificity of the peptide uponwhich the peptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery”, Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861).

A variety of peptidomimetics are known in the art and can be encompassedwithin embodiments described herein including, for example, peptide-likemolecules which contain a constrained amino acid, a non-peptidecomponent that mimics peptide secondary structure, or an amide bondisostere. A peptidomimetic that contains a constrained, non-naturallyoccurring amino acid can include, for example, an α-methylated aminoacid; α,α-dialkylglycine or α-aminocycloalkane carboxylic acid; anNα-Cacyclized amino acid; an Nα-methylated amino acid; αβ- or γ-aminocycloalkane carboxylic acid; an α,β-unsaturated amino acid; aβ,β-dimethyl or β-methyl amino acid; αβ-substituted-2,3-methano aminoacid; an N-Cδ or Cα-Cδcyclized amino acid; a substituted proline oranother amino acid mimetic. A peptidomimetic which mimics peptidesecondary structure can contain, for example, a nonpeptidic β-turnmimic; γ-turn mimic; mimic of β-sheet structure; or mimic of helicalstructure, each of which is well known in the art. A peptidomimetic alsocan be a peptide-like molecule which contains, for example, an amidebond isostere such as a retro-inverso modification; reduced amide bond;methylenethioether or methylene-sulfoxide bond; methylene ether bond;ethylene bond; thioamide bond; transolefin or fluoroolefin bond;1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylenebond or another amide isostere. One skilled in the art understands thatthese and other peptidomimetics are encompassed within the meaning ofthe term “peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptidedescribed herein, as well as potential geometrical and chemicalcomplementarity to a cognate receptor. Where no crystal structure of apeptide described herein is available, a structure can be generatedusing, for example, the program CONCORD (Rusinko et al., J. Chem. Inf.Comput. Sci. 29:251 (1989)). Another database, the Available ChemicalsDirectory (Molecular Design Limited, Informations Systems; San LeandroCalif.), contains about 100,000 compounds that are commerciallyavailable and also can be searched to identify potential peptidomimeticsof a peptide described herein, for example, having specificity for themicrobes.

Embodiments of Various Aspects Described Herein can be Defined in any ofthe Following Numbered Paragraphs:

-   1. A modified integrin α_(V) headpiece polypeptide comprising an    amino acid sequence of SEQ ID NO: 1 with at least one Cys residue    introduced thereto by one or more of the following modifications    (a)-(e):    -   a. substitution of amino acid residues 399-401 (Ser-Met-Pro)        with one of the following: (i) Ser-Cys-Pro; (ii)        Gly-Cys-Pro; (iii) Ser-Cys-Gly; (iv) Gly-Cys-Gly; (v)        Ser-Gly-Cys-Pro (SEQ ID NO: 59); (vi) Ser-Cys-Gly-Pro (SEQ ID        NO: 60); (vii) Gly-Cys-Gly-Pro (SEQ ID NO: 61); and (viii)        Ser-Gly-Cys-Gly (SEQ ID NO: 62);    -   b. substitution of amino acid residues 310-311 (Gln-Glu) with        Gly-Cys;    -   c. substitution of amino acid residues 299 (Leu) and 310 (Gln)        with Cys and Gly, respectively;    -   d. substitution of amino acid residues 302-311        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln-Glu) (SEQ ID NO: 63) with        Gly-Gln-Gly-Cys (SEQ ID NO: 64); and/or    -   e. substitution of amino acid residue 299 (Leu) to Cys and        substitution of amino acid residues 302-310        (Asp-Arg-Gly-Ser-Asp-Gly-Lys-Leu-Gln) (SEQ ID NO: 65) with        Gly-Gln-Gly.-   2. The modified integrin α_(V) headpiece polypeptide of paragraph 1,    wherein the modified integrin α_(V) headpiece polypeptide is a    soluble polypeptide.-   3. A modified integrin β₆ headpiece polypeptide comprising a βI    domain of integrin β₆ subunit with at least one Cys residue    introduced thereto by one or more of the following modifications    (f)-(h), the βI domain is defined from residues DYP to residues ELR    as shown in an amino acid sequence of SEQ ID NO: 2:    -   f. substitution of amino acid residue 270 (Ile) with Cys;    -   g. substitution of amino acid residue 294 (Thr) with Cys; and    -   h. substitution of amino acid residue 296 (Gly) with Cys.-   4. The modified integrin β₆ headpiece polypeptide of paragraph 3,    further comprising a PSI domain of integrin β₆, wherein the PSI    domain is defined from residues HVQ to residues NFI as shown in an    amino acid sequence of SEQ ID NO: 2.-   5. The modified integrin β₆ headpiece polypeptide of paragraph 3 or    4, further comprising a hybrid domain of integrin β₆, wherein the    hybrid domain is defined from residues ENP to residues QTE, and/or    from residues SEV to residues ECN as shown in an amino acid sequence    of SEQ ID NO: 2.-   6. The modified integrin β₆ headpiece polypeptide of any of    paragraphs 3-5, further comprising a EGF-1 domain of integrin β₆,    wherein the EGF-1 domain is defined from residues CDC to residues    SRG as shown in an amino acid sequence of SEQ ID NO: 2.-   7. The modified integrin β₆ headpiece polypeptide of paragraph 3,    which comprises an amino acid sequence of SEQ ID NO: 2 with said at    least one Cys residue introduced thereto by one or more the    modifications (f)-(h).-   8. The modified integrin β₆ headpiece polypeptide of any of    paragraphs 3-7, wherein the modified integrin β₆ headpiece    polypeptide is a soluble polypeptide.-   9. A modified integrin β₃ headpiece polypeptide comprising amino    acid residues 27 to 498 of SEQ ID NO: 5 with at least one Cys    residue introduced thereto by substitution of amino acid residue 293    (Gln) with Cys.-   10. The modified integrin β₃ headpiece polypeptide of paragraph 9,    wherein the modified integrin β₃ headpiece polypeptide is a soluble    polypeptide.-   11. A modified integrin β₈ headpiece polypeptide comprising amino    acid residues 43 to 498 of SEQ ID NO: 6 with at least one Cys    residue introduced thereto by substitution of amino acid residue 301    (Val) with Cys.-   12. The modified integrin s β₈ headpiece polypeptide of paragraph    11, wherein the modified integrin β₈ headpiece polypeptide is a    soluble polypeptide.-   13. A modified integrin polypeptide dimer comprising the modified    integrin α_(V) headpiece polypeptide of paragraph 1 or 2 and at    least one domain of an integrin β polypeptide selected from the    group consisting of a PSI domain, hybrid domain, βI domain, and an    EGF-1 domain, wherein the modified integrin α_(V) headpiece    polypeptide and said at least one domain of the integrin β    polypeptide are covalently linked by at least one disulfide bond.-   14. The modified integrin polypeptide dimer of paragraph 13, wherein    the modified integrin α_(V) headpiece polypeptide comprises    substitution of amino acid residues 399-401 (Ser-Met-Pro) with one    of the following: (a) Ser-Cys-Pro; (b) Gly-Cys-Pro; and (c)    Ser-Gly-Cys-Pro (SEQ ID NO: 59).-   15. The modified integrin polypeptide dimer of paragraph 13 or 14,    wherein the integrin β polypeptide is selected from the group    consisting of β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈.-   16. A modified integrin polypeptide dimer comprising an integrin α    polypeptide and the modified integrin β₆ headpiece polypeptide of    any of paragraphs 3-8, wherein the integrin α polypeptide and the    modified integrin β₆ headpiece polypeptide are covalently linked by    at least one disulfide bond.-   17. The modified integrin polypeptide dimer of paragraph 16, wherein    the modified integrin β₆ headpiece polypeptide comprises    substitution of amino acid residue 270 (Ile) with Cys.-   18. A modified integrin polypeptide dimer comprising an integrin α    polypeptide and the modified integrin β₃ headpiece polypeptide of    any of paragraphs 9-10, wherein the integrin α polypeptide and the    modified integrin β₃ headpiece polypeptide are covalently linked by    at least one disulfide bond.-   19. A modified integrin polypeptide dimer comprising an integrin α    polypeptide and the modified integrin β₈ headpiece polypeptide of    any of paragraphs 11-12, wherein the integrin α polypeptide and the    modified integrin β₈ headpiece polypeptide are covalently linked by    at least one disulfide bond.-   20. The modified integrin polypeptide dimer of any of paragraphs    16-19, wherein the integrin α polypeptide is selected from the group    consisting of α₁, α₂, α₃, α₄, α₅, α₆, α₇, α₈, α₉, α₁₀, α₁₁, α_(D),    α_(E), α_(L), α_(M), α_(V), α_(2B), and α_(X).-   21. The modified integrin polypeptide dimer of paragraph 20, wherein    the integrin α polypeptide is an integrin α_(V) polypeptide.-   22. A modified integrin polypeptide dimer comprising the modified    integrin α_(V) headpiece polypeptide of paragraph 1 or 2 and the    modified integrin β₆ headpiece polypeptide of any of paragraphs 3-8,    wherein the modified integrin α_(V) headpiece polypeptide and the    integrin β₆ headpiece polypeptide are covalently linked by at least    one disulfide bond.-   23. The modified integrin polypeptide dimer of paragraph 22, wherein    the modified integrin α_(V) headpiece polypeptide comprises    substitution of amino acid residues 399-401 (Ser-Met-Pro) with one    of the following: (i) Ser-Cys-Pro; (ii) Gly-Cys-Pro; and (iii)    Ser-Gly-Cys-Pro (SEQ ID NO: 59).-   24. The modified integrin polypeptide dimer of paragraph 22 or 23,    wherein the modified integrin β₆ headpiece polypeptide comprises    substitution of amino acid residue 270 (Ile) with Cys.-   25. A modified integrin polypeptide dimer comprising the modified    integrin α_(V) headpiece polypeptide of paragraph 1 or 2 and the    modified integrin β₃ headpiece polypeptide of any of paragraphs    9-10, wherein the modified integrin α_(V) headpiece polypeptide and    the integrin β₃ headpiece polypeptide are covalently linked by at    least one disulfide bond.-   26. The modified integrin polypeptide dimer of paragraph 25, wherein    the modified integrin α_(V) headpiece polypeptide comprises    substitution of amino acid residues 399-401 (Ser-Met-Pro) with (iii)    Ser-Gly-Cys-Pro (SEQ ID NO: 59).-   27. A modified integrin polypeptide dimer comprising the modified    integrin α_(V) headpiece polypeptide of paragraph 1 or 2 and the    modified integrin β₈ headpiece polypeptide of any of paragraphs    11-12, wherein the modified integrin α_(V) headpiece polypeptide and    the integrin β₈ headpiece polypeptide are covalently linked by at    least one disulfide bond.-   28. The modified integrin polypeptide dimer of paragraph 27, wherein    the modified integrin α_(V) headpiece polypeptide comprises    substitution of amino acid residues 399-401 (Ser-Met-Pro) with (iii)    Ser-Gly-Cys-Pro (SEQ ID NO: 59).-   29. A method for determining whether a test agent forms a complex    with an integrin, the method comprising contacting the modified    integrin polypeptide dimer of any of paragraphs 13-28 with the test    agent, and detecting formation of a complex comprising the modified    integrin polypeptide dimer and the test agent bound thereto, whereby    detection of a complex indicates that the test agent is capable of    forming a complex with the integrin.-   30. The method of paragraph 29, wherein the detecting comprises    crystallization of the complex.-   31. The method of paragraph 29 or 30, further comprising, prior to    the detecting, contacting the modified integrin polypeptide dimer    with a competing agent, wherein the competing agent is capable of    competing with the test agent to bind the modified integrin    polypeptide dimer.-   32. The method of paragraph 31, wherein the competing agent is a    competing peptide.-   33. The method of paragraph 32, wherein the competing peptide    comprises an amino acid sequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu/Ile    (SEQ ID NO: 79), wherein X₁, X₂, and X₃ are each independently an    amino acid molecule.-   34. The method of paragraph 33, wherein the X₁ and/or X₃ is a Gly    molecule.-   35. The method of paragraph 33 or 34, wherein the X₂ is an Arg    molecule.-   36. The method of any of paragraphs 29-35, wherein the competing    agent comprises a detectable label.-   37. The method of paragraphs 29 or 30, wherein the test agent    comprises a detectable label.-   38. The method of paragraph 36 or 37, wherein the detectable label    comprises biotin, a fluorescent dye or molecule, a luminescent or    bioluminescent marker, a radiolabel, an enzyme, a quantum dot, an    imaging agent, or any combination thereof.-   39. The method of paragraph 38, wherein the detectable label    comprises a fluorescent molecule.-   40. The method of paragraph 39, wherein the detecting is performed    by fluorescence anisotropy and/or flow cytometry.-   41. A method for determining binding affinity of a test agent to an    integrin, the method comprising (i) contacting the modified integrin    polypeptide dimer of any of paragraphs 13-28 with the test agent and    a competing agent, wherein the competing agent comprises a    detectable label and is capable of competing with the test agent to    bind the modified integrin polypeptide dimer; and (ii) detecting a    signal from the detectable label of the competing agent that forms a    complex with the integrin, whereby a decrease in the detected signal    relative to a signal corresponding to saturation binding of the    competing agent to the modified integrin polypeptide dimer indicates    that the test agent has a higher binding affinity than the competing    agent to the integrin.-   42. The method of paragraph 41, wherein the competing agent is a    competing peptide.-   43. The method of paragraph 42, wherein the competing peptide    comprises an amino acid sequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu    (SEQ ID NO: 66), wherein X₁, X₂, and X₃ are each independently an    amino acid molecule.-   44. The method of paragraph 43, wherein the X₁ and/or X₃ is a Gly    molecule.-   45. The method of paragraph 43 or 44, wherein the X₂ is an Arg    molecule.-   46. The method of any of paragraphs 41-45, wherein the detecting    comprises crystallization of the complex.-   47. The method of any of paragraphs 41-46, wherein the detectable    label comprises biotin, a fluorescent dye or molecule, a luminescent    or bioluminescent marker, a radiolabel, an enzyme, a quantum dot, an    imaging agent, or any combination thereof.-   48. The method of paragraph 47, wherein the detectable label    comprises a fluorescent molecule.-   49. The method of paragraph 48, wherein the detecting is performed    by fluorescence anisotropy and/or flow cytometry.-   50. The method of any of paragraphs 41-49, wherein the    concentrations of the test agent and the competing agent are    essentially the same.-   51. A method of identifying an anti-α_(V)β₆ inhibitor comprising:    -   (a) generating on a computer a molecular representation of a        pharmacophore comprising a basic functional group, an acidic        functional group for coordination of a metal ion to a metal        ion-dependent adhesion site (MIDAS) in integrin β6 subunit, a        first hydrophobic functional group, and a second hydrophobic        functional group, wherein the functional groups are arranged to        satisfy the following conditions:        -   the distance between the first hydrophobic functional group            (H1) and the second hydrophobic functional group (H2) is            about 7-8 Å; the distance between the second hydrophobic            functional group (H2) and the basic functional group (B) is            about 8-9 Å; the distance between the basic functional            group (B) and the acidic functional group (A) is about 15-16            Å; the distance between the first hydrophobic functional            group (H1) and the acidic functional group (A) is about            14.5-15.5 Å; and the distance between the second hydrophobic            functional group (H2) and the acidic functional group (A) is            about 19-20 Å; and        -   the angle formed by H1-A-B is about 20°-24°; the angle            formed by H1-A-H2 is about 17°-21°; the angle formed by            H2-A-B is about 26°-30°; the angle formed by A-B-H1 is about            68°-72°; the angle formed by A-B-H2 is about 96°-100°; and            the angle formed by H1-B-H2 is about 49°-53°;    -   (b) generating on a computer atomic coordinates of an α_(V)β₆        integrin protein or a portion thereof having at least a        hydrophobic binding pocket in β₆ subunit; and    -   (c) determining on a computer likelihood of the molecular        representation interacting with one or more residues of the        computer-generated α_(V)β₆ integrin protein or a portion        thereof, thereby identifying a candidate anti-α_(V)β₆ inhibitor.-   52. The method of paragraph 37, wherein the first and second    hydrophobic functional groups are each independently selected from    an aromatic ring (aryl) or a linear moiety.-   53. A method of identifying an anti-α_(V)β₆ inhibitor comprising:    -   (a) providing, on a computer, a three-dimensional crystalline        structure of α_(V)β₆ integrin protein or a portion thereof        characterized by atomic structure coordinates (e.g., as        described in Table 6 of PCT Application No: PCT/US15/44093 filed        Aug. 6, 2015), or a three-dimensional structure that exhibits a        root-mean-square difference (rmsd) in α-carbon positions of less        than 2.0 Å (or less than 1.0 Å) with the atomic structure        coordinates (e.g., described in Table 6 PCT Application No:        PCT/US15/44093 filed Aug. 6, 2015);    -   (b) docking on the computer a first molecular entity in a first        part of a binding pocket of the integrin β₆ headpiece        polypeptide having an amino acid sequence of SEQ ID NO: 2, to        determine the binding association between the first molecular        entity and the first part of the binding pocket, wherein the        binding pocket comprises amino acid residues Ala-217, Asn-218,        Pro-179, Cys-180, Ile-183, Ala-126, and Tyr-185;    -   (c) docking on the computer a second molecular entity in a        second part of the binding pocket, to determine the binding        association between the second molecular entity and the second        part of the binding pocket;    -   (d) repeating steps (b) to (c) with a different first and second        molecular entity;    -   (e) selecting a first and a second chemical entity based on the        quantified binding associations; and    -   (f) generating on the computer a pharmacophore model by        assembling the selected first and second molecular entity into a        molecular representation that interacts with the binding pocket.-   54. The method of any of paragraphs 37-39, further comprising    contacting the candidate anti-α_(V)β₆ inhibitor (based on the    pharmacophore model) with an α_(V)β₆ integrin protein to determine    the ability of the candidate anti-α_(V)β₆ integrin inhibitor to bind    the α_(V)β₆ integrin protein.-   55. A crystalline composition comprising a ligand-binding headpiece    of integrin α_(V)β₆, wherein the crystalline composition is    characterized with space group C2, and has unit cell parameters    a=184.5±3 Å, b=168.3±3 Å, c=101.8±3 Å, α=β=90°, and γ=98.2°±3°.-   56. A crystalline composition comprising a ligand-binding headpiece    of integrin α_(V)β₆ and a liagnd, wherein the crystalline    composition is characterized with space group C2, and has unit cell    parameters a=184.4±3 Å, b=170.0±3 Å, c=102.4±3 Å, α=β=90°, and    γ=98.7°±3°.-   57. The crystalline composition of paragraph 41 or 42, wherein the    ligand-binding headpiece of integrin α_(V)β₆ comprises a modified    integrin α_(V) headpiece polypeptide of paragraph 1, and a modified    integrin β₆ headpiece polypeptide of paragraph 3.-   58. The crystalline composition of paragraph 43, wherein the    crystalline composition has a binding pocket in the modified    integrin β₆ headpiece polypeptide, wherein the binding pocket    comprises amino acid residues Ala-217, Asn-218, Pro-179, Cys-180,    Ile-183, Ala-126, and Tyr-185.-   59. The crystalline composition of any of paragraphs 41-44, wherein    the ligand-binding headpiece of integrin α_(V)β₆ is described by the    atomic structure coordinates (e.g., described in Table 6 of PCT    Application No: PCT/US15/44093 filed Aug. 6, 2015), or a structure    that exhibits a root-mean-square difference (rmsd) in α-carbon    positions of less than 2.0 Å (or less than 1.0 Å) with the atomic    structure coordinates (e.g., described in Table 6 of PCT Application    No: PCT/US15/44093 filed Aug. 6, 2015).-   60. The crystalline composition of any of paragraphs 41-45, wherein    the crystal composition is formed from a crystallization solution    buffered between pH 6-8 at a temperature of about 20° C. and having    an ionic strength of about 800-900 mM.-   61. A method of identifying an anti-α_(V)β₃ inhibitor comprising:    -   (a) providing, on a computer, a three-dimensional crystalline        structure of α_(V)β₃ integrin protein or a portion thereof        characterized by atomic structure coordinates(e.g., as described        in Table 8 of PCT Application No: PCT/US15/44093 filed Aug. 6,        2015), or a three-dimensional structure that exhibits a        root-mean-square difference (rmsd) in α-carbon positions of less        than 2.5 Å (or less than 2.0 Å, or less than 1.0 Å) with the        atomic structure coordinates(e.g., as described in Table 8 of        PCT Application No: PCT/US15/44093 filed Aug. 6, 2015);    -   (b) docking on the computer a first molecular entity in a first        part of a binding pocket of the integrin β₃ headpiece        polypeptide having an amino acid sequence of SEQ ID NO: 5 or a        fragment thereof, to determine the binding association between        the first molecular entity and the first part of the binding        pocket;    -   (c) docking on the computer a second molecular entity in a        second part of the binding pocket, to determine the binding        association between the second molecular entity and the second        part of the binding pocket;    -   (d) repeating steps (b) to (c) with a different first and second        molecular entity;    -   (e) selecting a first and a second chemical entity based on the        quantified binding associations; and    -   (f) generating on the computer a pharmacophore model by        assembling the selected first and second molecular entity into a        molecular representation that interacts with the binding pocket.-   62. The method of paragraph 61, further comprising contacting the    candidate anti-α_(V)β₃ inhibitor (based on the pharmacophore model)    with an α_(V)β₃ integrin protein to determine the ability of the    candidate anti-α_(V)β₃ integrin inhibitor to bind the α_(V)β₃    integrin protein.-   63. A crystalline composition comprising a ligand-binding headpiece    of integrin α_(V)β₃, wherein the crystalline composition is    characterized with space group P22₁2₁, and has unit cell parameters    a=87±2 Å, b=124±2 Å, c=165±2 Å, α=β=90°, and γ=90°±3°.-   64. The crystalline composition of paragraph 63, wherein the    ligand-binding headpiece of integrin α_(V)β₃ comprises a modified    integrin α_(V) headpiece polypeptide of paragraph 1, and a modified    integrin β₃ headpiece polypeptide of paragraph 9.-   65. The crystalline composition of any of paragraphs 63-64, wherein    the ligand-binding headpiece of integrin α_(V)β₃ is described by the    atomic structure coordinates(e.g., as described in Table 8 of PCT    Application No: PCT/US15/44093 filed Aug. 6, 2015), or a structure    that exhibits a root-mean-square difference (rmsd) in α-carbon    positions of less than 2.5 Å (or less than 2.0 Å, or less than 1.0    Å) with the atomic structure coordinates(e.g, described in Table 8    of PCT Application No: PCT/US15/44093 filed Aug. 6, 2015).-   66. The crystalline composition of any of paragraphs 63-65, wherein    the crystal composition is formed from a crystallization solution    buffered between pH 6-8 at a temperature of about 20° C. and having    an ionic strength of about 800-900 mM.-   67. A crystalline composition comprising a ligand-binding headpiece    of integrin α_(V)β₈, wherein the crystalline composition is    characterized with space group P1, and has unit cell parameters    a=153±3 Å, b=55±3 Å, c=181±3 Å, α=β=90°, and γ=110°±3°.-   68. The crystalline composition of paragraph 67, wherein the    ligand-binding headpiece of integrin α_(V)β₈ comprises a modified    integrin α_(V) headpiece polypeptide of paragraph 1, and a modified    integrin β₈ headpiece polypeptide of paragraph 11.-   69. The crystalline composition of any of paragraphs 67-68, wherein    the crystal composition is formed from a crystallization solution    buffered between pH 6-8 at a temperature of about 20° C. and having    an ionic strength of about 800-900 mM.-   70. A method of identifying an anti-α_(V)β₈ inhibitor comprising:    -   (a) providing, on a computer, a three-dimensional crystalline        structure of α_(V)β₈ integrin protein or a portion thereof        derived from the crystalline composition of any of paragraphs        67-69;    -   (b) docking on the computer a first molecular entity in a first        part of a binding pocket of the integrin β₈ headpiece        polypeptide having an amino acid sequence of SEQ ID NO: 6 or a        fragment thereof, to determine the binding association between        the first molecular entity and the first part of the binding        pocket;    -   (c) docking on the computer a second molecular entity in a        second part of the binding pocket, to determine the binding        association between the second molecular entity and the second        part of the binding pocket;    -   (d) repeating steps (b) to (c) with a different first and second        molecular entity;    -   (e) selecting a first and a second chemical entity based on the        quantified binding associations; and    -   (f) generating on the computer a pharmacophore model by        assembling the selected first and second molecular entity into a        molecular representation that interacts with the binding pocket.-   71. The method of paragraph 70, further comprising contacting the    candidate anti-α_(V)β₈ inhibitor (based on the pharmacophore model)    with an α_(V)β₈ integrin protein to determine the ability of the    candidate anti-α_(V)β₈ integrin inhibitor to bind the α_(V)β₈    integrin protein.-   72. A modified integrin α_(V) headpiece polypeptide comprising,    essentially consisting of, or consisting of an amino acid sequence    of SEQ ID NO: 131.-   73. A modified integrin β₆ headpiece polypeptide comprising an amino    acid sequence of SEQ ID NO: 133 (with or without a His₆ tag (SEQ ID    NO: 132)) or SEQ ID NO: 135.-   74. A modified integrin β₃ headpiece polypeptide comprising,    essentially consisting of, or consisting of an amino acid sequence    of SEQ ID NO: 136.-   75. A modified integrin β₈ headpiece polypeptide comprising,    essentially consisting of, or consisting of an amino acid sequence    of SEQ ID NO: 134 or SEQ ID NO: 137.-   76. A modified integrin polypeptide dimer comprising, essentially    consisting of, or consisting of (i) the modified integrin α_(V)    headpiece polypeptide of paragraph 72, and (ii) the modified    integrin β₆ headpiece polypeptide of paragraph 73.-   77. A modified integrin polypeptide dimer comprising, essentially    consisting of, or consisting of (i) the modified integrin α_(V)    headpiece polypeptide of paragraph 72, and (ii) the modified    integrin β₃ headpiece polypeptide of paragraph 74.-   78. A modified integrin polypeptide dimer comprising, essentially    consisting of, or consisting of (i) the modified integrin α_(V)    headpiece polypeptide of paragraph 72, and (ii) the modified    integrin β₈ headpiece polypeptide of paragraph 75.

As used herein, the term “comprising” or “comprise(s)” is used inreference to compositions, methods, and respective component(s) thereof,that are essential to the invention, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein, the term “consisting essentially of” or “consist(s)essentially of” or “essentially consisting of,” or “essentiallyconsist(s) of” refers to those elements required for a given embodiment.The term permits the presence of additional elements that do notmaterially affect the basic and novel or functional characteristic(s) ofthat embodiment of the invention.

As used herein, the term “consisting of” or “consist(s) of” refers tocompositions, methods, and respective components thereof as describedherein, which are exclusive of any element not recited in thatdescription of the embodiment.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1. Example Methods of Expression, Affinity Measurement, andInhibitor Pharmacophore Model of Integrin α_(V)β₆

As integrins are non-covalently linked heterodimers of α and β subunits,to prevent α/β dissociation, a disulfide bond was introduced tocovalently link the subunits together. A series of cysteine combinationswas designed to form the disulfides, based on the crystal structure ofintegrin α_(V)β₆. Residues in the α_(V) β-propeller domain and β₆βI-domain, which are distal from the ligand-binding site, wereevaluated. Twelve α_(V) mutants and three β₆ mutants chosen are listedbelow:

On α_(V), residues S399-M400-P401 (SMP) are mutated as follows,including some mutations that insert an extra residue: α_(V)s1. SCP,α_(V) β₆s2. GCP, α_(V)s3. SCG, α_(V)s4. GCG, α_(V)s5. SGCP (SEQ ID NO:59), α_(V)s6. SCGP (SEQ ID NO: 60), α_(V)s7. GCGP (SEQ ID NO: 61),α_(V)s8. SGCG (SEQ ID NO: 62).

Other mutations in α_(V) are:

-   -   α_(V)s9. Q310 and E311 to G310 and C311 (Q310G and E311C)    -   α_(V)s10. L299 and Q310 to C299 and G310 (L299C and Q310G)    -   α_(V)s11. 302DRGSDGKLQE311 (SEQ ID NO: 63) to GQGC (SEQ ID        NO: 64) (this involves deletion of a loop)    -   α_(V)s12. L299 to C299 (L299C) plus 302DRGSDGKLQ310 (SEQ ID        NO: 65) to GQG

On β₆, the mutations are:

-   -   β₆s1. 1270 to C270 (I270C)    -   β₆s2. T294 to C294 (T294C)    -   β₆s3. G296 to C296 (G296C)

Expression tests of the mutant combinations were performed in 293s GnTI⁻cells. Two days after transient transfection, supernatants weresubjected to western blot using anti-his antibody. Results showed thatα_(V)s1, α_(V)s2, or α_(V)s5 with β₆s1 could be highly expressed underthe experiment settings, among which, the combination of α_(V)s5/β₆s1showed the highest expression level.

It was next sought to determine whether a disulfide bridge can beintroduced to other integrin heterodimers. α₅β₁, α_(V)β₃, α_(V)β₈, α₄β₁and α₄β₇. Results showed that α₅β₁, α_(V)β₃, and α_(V)β₈ can form thedisulfide bond, whereas α₄β₁ and α₄β₇ cannot.

Example 2. Binding Affinity Measurements by Fluorescence Anisotropy

A fluorescence probe for integrin α_(V)β₆, the sequence of which isFITC-GRGDLGRL (SEQ ID NO: 68), was synthesized. Saturation bindingaffinity measurements showed the probe binds to α_(V)β₆ at 100 nM and 10nM under physiological buffer containing 1 mM Mg²⁺/Ca²⁺ or Mn²⁺/Ca²⁺,respectively. By competition binding assay, the affinities of differentligands were measured.

Example 3. Pharmacophore Model for Anti-α_(V)β₆ Inhibitor

According to the crystal structure of α_(V)β₆ in complex with TGF-β3peptide (e.g., as described in Example, a pharmacophore model for ananti-α_(V)β₆ inhibitor was built. The pharmacophore comprises threefeatures, a basic functional group, an acidic functional group forcoordination of the metal ion to the MIDAS in β₆, and two hydrophobicfunctional groups.

Example 4. Example Use of the Modified Integrin Polypeptide DimersDescribed Herein—to Identify Structural Determinants of Integrinβ-Subunit Specificity for Latent TGF-β

Eight integrin α/β heterodimers including five with the α_(V) subunitrecognize ligands with an Arg-Gly-Asp (RGD) motif. However, thestructural mechanism by which integrins differentiate among the manyextracellular proteins with RGD motifs and achieve specificity is notunderstood. In this example, how α_(V)β₆ and α_(V)β₈, which are uniquelyimportant in the activation of TGF-β in vivo, achieve specificity wasinvestigated. As presented herein, TGF-β activation by α_(V)β₆ andα_(V)β₈ is related to unusually high affinity. Crystal structures showthe determinants of the high affinity of α_(V)β₆ for a pro-TGF-β3undecapeptide and mutations extend the results to activation ofpro-TGF-β1. Both an RGD motif and a following LXXL/I motif that foldsinto an amphipathic α-helix when bound to the integrin β-subunit arerequired for high affinity. Structural elucidation of the basis forligand-binding specificity by the integrin β-subunit indicatescontributions by three different βI domain loops, which we propose todesignate specificity-determining loop (SDL)-1, 2, and 3. Variation in apair of single key residues in SDL-1 and 3 correlates with the variationof the entire β subunit in integrin evolution.

Pro-TGF-β1 Activation by Integrin Correlates with High Affinity

Transfectants expressing α_(V)β₆ and α_(V)β₈, but not α_(V)co-transfected with the β₁, β₃ and β₅ subunits, can activate pro-TGF-β(FIG. 1A). Correlating with activation, α_(V)β₆ and α_(V)β₈, but notother α_(V) integrin transfectants, strongly bound 50 nM FITC-pro-TGF-β1(FIG. 1B).

Ligands bind to the integrin headpiece, which contains the α-subunitβ-propeller domain and thigh domain, and the β-subunit βI, hybrid, PSI,and EGF1 domain. Using competition with the pro-TGF-β3 peptideFITC-GRGDLGRL (SEQ ID NO: 68), peptide affinity for the α_(V)β₆ andα_(V)β₃ headpiece was measured with fluorescence anisotropy.Nonapeptides containing RGD from pro-TGF-β1 and pro-TGF-β3 bound toα_(V)β₆ with remarkably high affinity of 10.3 nM and 8.5 nM (FIG. 1C).In contrast, the same peptides bound to α_(V)β₃ with 1,000-fold loweraffinity (FIG. 1D). Interestingly, the homologous peptide frompro-TGF-β2, which has SGDQ (SEQ ID NO: 70) in place of RGDL (SEQ ID NO:71) of pro-TGF-β1, also bound to α_(V)β₆, but with 1,000-fold loweraffinity (8.5 μM), and was comparable in affinity to the GRGDSP (SEQ IDNO: 72) peptide of fibronectin (2.5 μM) (FIG. 1C).

Example α_(V)β₆ Crystal Structures

α_(V)β₆ crystal structures (e.g., one or more embodiments of themodified integrin polypeptide dimers described herein) were used todetermine the basis for the high affinity of α_(V)β₆ for pro-TGF-β andits peptides. Crystals of the α_(V)β₆ headpiece, with or without apro-TGF-β3 undecapeptide soaked therein, diffracted to 2.5 and 2.85 Årespectively, and contain two molecules per asymmetric unit with almostidentical structures. Table 4 below shows statistics of X-raydiffraction and structure refinement.

αvβ6 αvβ6 + TGF-β3 peptide Data collection statistics Space Group C2 C2α, β, γ ° 90, 90, 98.2 90, 90, 98.7 Unit Cell (a, b, c) Å 184.5, 168.3,101.8 184.4, 170.0, 102.4 Resolution range (Å) 50.0-2.85 (2.92)-2.85)^(a) 50.0-2.50 (2.67-2.50) Compeleteness (%) 97.4 (91.0) 94.4 (70.6)Number unique reflections 69,928 (4,837) 87,425 (5,641) Redundancy 2.4(2.3) 2.3 (2.1) R_(merge) (%) ^(b) 17.5 (349) 11.6 (233) I/σ(I) 4.8(0.33) 4.9 (0.35) CC_(1/2) (%)^(c) 98.2 (10.4) 98.3 (10.4) Wavelength(Å) 1.0332 1.0332 Refinement Statistics R_(work) (%)^(d) 23.6 (38.0)22.2 (39.9) R_(free) (%) 27.9 (41.7) 26.6 (44.4) Bond RMSD (Å) 0.0050.009 Angle RMSD (°) 0.76 1.2 Ramachandran plot^(c) 95.7/4.1/0.295.8/4.0/0.2 (Favored/allowed/outlier) Number of waters 161 207 PDB 4UM84UM9 ^(a) The numbers in parentheses refer to the highest resolutionshell. ^(b) Rmerge = Σh Σi |Ii(h) − <I(h)> |/ΣhΣi Ii(h), where Ii(h) and<I(h)> are the i^(th) and mean measurement of the intensity ofreflection h. ^(c)Pearson's correlation coefficient between averageintensities of random half-datasets for each unique reflection (25).^(d)Rfactor = Σh||Fobs (h)|−|Fcalc (h)||/Σh|Fobs (h)|, where Fobs (h)and F calc (h) are the observed and calculated structure factors,respectively. No I/σ(I) cutoff was applied. ^(e)Calculated withMolProbity (28).

The headpiece adopts the closed headpiece conformation in the absence ofligand (FIG. 2A), i.e. with the hybrid domain swung in toward theα-subunit and with the β1-α1, and β6-α7 loops and α7-helix in the βIdomain in the closed conformation (8, 12-14). Compared to β₃, the β₆ βIand PSI domains are similar in structure, with greater differences inthe hybrid domain (Table 5).

TABLE 5 Cα Root-Mean-Square Deviation (RMSD) to α_(V)β₆ headpiece-TGF-βpeptide complex (RMSD was calculated using the align command of PyMolwith zero refinement cycles.) Unliganded α_(V)β₆ α_(V)β₃ (3G1E)α_(IIb)β₃ (2FCS) Domain (Å) (Å) (Å) β-propeller 0.3 0.5 2.8 Thigh 0.40.9 2.6 βI 0.6 0.9 0.9 Hybrid 0.6 1.5 1.8 PSI 0.5 0.8 0.9 EGF1 NA 3.23.2

Three closely spaced metal ion binding sites are present in the integrinβI domain, the synergistic metal ion binding site (SyMBS), metal iondependent adhesion site (MIDAS), and adjacent to MIDAS (ADMIDAS).α_(V)β₆ crystallized at pH 6.5 loses its SyMBS metal ion; furthermore,the SyMBS-coordinating α2-α3 loop also remodels and invades theligand-binding pocket (FIG. 2B). Remodeling enables SyMBS residuesAsn-218 and Asp-220 to point outward and form three strong, 2.4 to 2.7 Åhydrogen bonds in place of Ca²⁺-coordination (FIG. 2B). Similarremodeling of the β₃-subunit α2-α3 loop in the absence of a SyMBS Ca²⁺is blocked by the large sidechains that characterize its ligand bindingpocket, especially β₃ Arg-214 and Tyr-166 in place of β₆ Ala-217 andLys-170 (FIG. 2F-2H).

The inventors hypothesized that crystallization at pH 4.5-6.5 might beresponsible for variable loss of SyMBS, MIDAS, and/or ADMIDAS metal ionsfrom α_(V)β₃ (12, 14) and α_(V)β₆, in contrast to occupation of allthree sites in α_(IIB)β₃ crystalized at higher pH (13, 15). To evaluatethis hypothesis, the effect of pH on affinity of α_(V)β₆ for the TGF-β3nonapeptide was examined. Fluorescence anisotropy demonstrated strong pHdependence with a sharp decrease in affinity between pH 7 and 6 (FIG.2C). As many cells co-express integrins with their ligands, includingepithelial cells that coexpress α_(V)β₆ and pro-TGF-βI, without wishingto be bound by theory, this pH-dependence may contribute to theinhibition of ligand binding during biosynthesis in the Golgi (pH6.0-6.7) and transport in endosomes (pH 6.3-6.5) (16).

Ligand Binding by α_(V)β₆

Soaking ligand into α_(V)β₆ crystals restored a Ca²⁺-bound conformationof the SyNMBS α2-α3 loop (FIG. 2B) and showed how TGF-β3 peptide bindswith high affinity (FIG. 2D). Ligand binding induced a local ˜1.5 Ådisplacement of the β1-α1 loop toward the Asp of RGD and the MIDAS Mg²⁺(FIG. 2B), as seen in intermediate states of other integrins with RGDsoaked in (9, 12, 15). The Asp of RGD coordinated the MIDAS Mg²⁺ ionthrough one sidechain oxygen and formed hydrogen bonds to NH groups ofAsn-218 and Ala-126 through the other sidechain oxygen (FIG. 2D). TheArg of RGD formed bidentate hydrogen bonds through its guanido group tothe sidechain of Asp-218 in the α_(V) β-propeller domain (FIG. 2D), asin binding to α_(V)β₃ (FIG. 2E) (7). Furthermore, as the ligand spannedthe α_(V)-β₆ interface, the backbone of the RGD Arg formed a hydrogenbond to the sidechain of Thr-221 in the β₆ α2-α3 loop (FIG. 2D). Asimilar hydrogen bond to the ligand backbone can form with β₈, but notwith β₃ or β₅, which have Ala in the position of β₆ Thr-221 (FIG. 2E andFIG. 2H).

The largest conformational difference in the ligand-binding regionbetween α_(V) β₆ and α_(V) β₃ is in the β2-β3 loop. This loop isdisplaced in β₆ relative to β₃ both as a consequence of sequencedifferences in the β2-β3 loop itself, and in the β1-α1 and α2-α3 loopswith which it interacts (FIGS. 2F-2H). The path of the β2-β3 loop isaltered in β3 by the insertion of cis-Pro-169 (FIG. 2G and FIG. 2H) aswell as π-cation bonds between β2-β3 residue Tyr-166 and α2-α3 residuesArg-214 and Arg-216 (FIG. 2G). The three residues forming π-cation bondsare replaced in β₆ by Lys-170, Ala-217 and Ile-219 (FIG. 2F).Furthermore, a hydrogen bond between Tyr-185 in the β2-β3 loop andAsp-129 in the β1-α1 loop constrains the conformation at the C-terminalportion of the β2-β3 loop in β₆ (FIG. 2F). Thus, backbone differences inthe β2-β3 loop derive not only from the difference in its own sequence,but also from differences in sequences of loops that interact with theβ2-β3 loop.

Strikingly, the TGF-β3 peptide forms an α-helix which extensivelyinterfaces with the β₆-subunit (FIG. 2A and FIG. 2D). Immediatelyfollowing the Asp of RGD, the sequence L²⁴⁴GRL²⁴⁷K (SEQ ID NO: 73) formsan amphipathic α-helix. TGF-β Leu-244 binds in a β₆-subunit hydrophobicpocket formed by the sidechain of Ala-217 and backbone of Asn-218 in theα2-α3 loop, the backbone of Pro-179 and sidechains of Cys-180 andIle-183 in the β2-β3 loop, and sidechain of Ala-126 in the β1-α1 loop(FIG. 2D). The aliphatic portion of the ligand Lys-248 sidechaincontributes to burying Leu-244. Ligand residue Leu-247 further buriesLeu-244, and binds in the same hydrophobic pocket by interacting withthe backbone and sidechain of Ala-126 in the β1-α1 loop; and with thesidechain of Ile-183, the disulfide bond of Cys-177 and Cys-184, and thearomatic ring of Tyr-185 in the β2-β3 loop. Thus, three different loopsin the βI domain make contacts with TGF-β ligand (FIG. 2D).

Integrin β-subunits vary markedly at the positions in the β1-α1 andα2-α3 loops where Ala-126 and Ala-217 contact the amphipathic TGF-βα-helix (FIG. 2H). Ala-126, in the β₆ DLSA¹²⁶S (SEQ ID NO: 74) MIDASmotif, is conserved as Ala in β₈, but is a Tyr in β₃ (Tyr-122, FIG. 2G)and in the β₁, β₂, and β₇ subunits (FIG. 2H). Introduction of the Tyrresidue with the A126Y mutation significantly decreased both binding ofpro-TGF-β1 and activation of TGF-β1 (FIG. 1A and FIG. 1B). Ala-217 inthe α2-α3 loop is a small residue (Gly or Ala) in most integrinβ-subunits, but a large Arg in the β₃ and β₅ subunits (FIG. 2H). TheA217R and double A126Y/A217R mutations completely abolished pro-TGF-β1binding and activation (FIG. 1A and FIG. 1B).

Between the two disulfide-bonded cysteines in the β2-β3 loop, where I183and Y185 contact the pro-TGF-β α-helix, integrins are highly diverse (6)(FIG. 2H). However, individual β₆ I183Y and Y185S mutations had noeffect, and the double mutation only slightly affected α_(V)β₆ bindingand activation of pro-TGF-β1 (FIG. 1A and FIG. 1B).

The Integrin-Binding Loop in Pro-TGF-β

In the ligand, the Gly residue preceding RGD extends back towards theamphipathic α-helix (FIG. 2D). Thus, Gly-240 and Lys-248 inG²⁴⁰RGDLGRLK²⁴⁸ (SEQ ID NO: 75) are only 8 Å apart (FIG. 2D). Thesequence in between has an overall loop-like conformation, with Asp-243and Leu-244 most buried in the binding pocket, which is centered on theβ₆ subunit rather than the α_(V)β₆ interface (FIG. 2D). Because the twoends of the pro-TGF-β3 peptide are near one another and orient away fromthe integrin, the peptide complex is highly compatible with integrinbinding to a pro-TGF-β3 macromolecule.

The integrin-binding residues identified in this Example lie near themiddle of an 18-residue segment that is disordered or only weaklyordered in the structure of pro-TGF-β1 (17), and can easily protrudefrom the shoulder region of pro-TGF-β to bind α_(V)β₆ in the helicalconformation identified in this Example. Interestingly, FIG. 3A showsthat foot-and-mouth disease virus (FMDV) utilizes an RGD motif followedby an amphipathic sequence very similar to those in pro-TGF-β1 and β3(18) to bind integrin α_(V) β₆ and infect epithelial cells.

The importance of the amphipathic α-helix for binding to α_(V) β₆ wasevaluated by helix truncation and mutation. The C-terminus of the TGF-β3undecapeptide was truncated one residue at a time (FIG. 3B). The largeststep-wise drops in affinity, about 10-fold each, occurred when Leu-244and then Leu-247 were removed (FIG. 3B). To disrupt the α-helixconformation upon binding of pro-TGF-β1 to α_(V) β₆, residues Ala-220and Thr-221, which correspond to α-helix residues Gly-245 and Arg-246 inthe pro-TGF-β3 peptide and have no contact with α_(V)β₆, were mutated toproline. These mutations had no effect on pro-TGF-β1 expression (FIG.3C), consistent with the postulated lack of importance of this regionfor structural stability. However, the double Pro mutant was deficientin ability to be activated by α_(V)β₆ (FIG. 3E), indicating theimportance of the α-helical conformation for interaction withpro-TGF-β1.

Specificity Determinants of Integrin β Subunits

The inventors have showed specializations in an integrin β-subunit thatenable ligand recognition with high affinity and specificity. α_(V)β₆not only recognizes RGD, but also a LXXL/I motif that folds into anamphipathic α-helix fitting into a hydrophobic pocket composed solely ofresidues from the β₆ subunit. In contrast, complexes of α_(V)β₃,α_(IIb)β₃, and α₅β₁ have revealed little interaction beyond that withRGD itself (7, 9, 19). The β₆ hydrophobic pocket interaction with theamphipathic α-helix as shown in this Example enables α_(V)β₆ to achieve˜1,000-fold selectivity for pro-TGF-β over the RGD motif present infibronectin and ˜1000-fold selectivity over α_(V)β₃ for the recognitionof pro-TGFβ.

Previously, little has been known about the contribution of integrin βsubunits to ligand recognition beyond the MIDAS. The inventors presentedherein that high affinity of α_(V)β₆ for pro-TGF-β is contributed by theβI domain β1-α1, β2-β3, and α2-α3 loops, each of which interacts in acrystal structure with an amphipathic α-helix in the ligand. These loopsare designated as the specificity-determining loops (SDL) 1, 2, and 3,according to their order in the amino acid sequence (FIGS. 2D-2H).Consistent with the structural observations on α_(V)β₆ bound topro-TGF-β3 peptide described herein, the inventors demonstrated withmutations that SDL1 (α1-α1 loop) and SDL3 (α2-α3 loop) are importantboth for binding to pro-TGF-β1 and for its activation. SDL2 (β2-β3 loop)has previously been shown by mutation to be important for ligandselectivity by α_(V)β₃ and α_(V)β₁ (6).

In addition to contacting the amphipathic helix, the β1-α1 (SDL1) andα2-α3 (SDL3) loops contact the Gly and Asp of RGD. The SDL designationfor the three loops in the βI domain that bind ligand is analogous tothe complementarity determining region (CDR) designation for antibodyand T cell receptor chains, which also use three surface-exposed loopsto contact ligand.

SDL1 and SDL3 have the sequences D(L/V/F)SX₁SMX₂(D/N)(D/N) (SEQ ID NO:76) and (V/I)SX₁NX₂D(A/S/T)PE (SEQ ID NO: 77), respectively (FIG. 2H).The two alanines in β₆ that contact pro-TGF-β are in the X₁ position ineach loop. Variation in the X₁ positions of the β1-α1 and α2-α3 loopscorrelates with variation in the entire β subunit in evolution (FIG. 2H)(20), indicating a paradigmatic role in overall β subunit function.Thus, the β₆ and β₈ subfamily (A in β1-α1+A/G in β2-β3), the β₁, β₂, andβ₇ subfamily (Y+G), the β₃ and β₅ subfamily (Y/L+R), and β₄ (N+G) eachhave a unique pattern, and within a subfamily, variation is confined tochemically similar residues (A/G and Y/L). Residues in the X₂ positionin SDL1 and SDL3 pack against and contribute to conformational variationof SDL2 (FIGS. 2F-2H). SDL1 and SDL3 also bind metal ions (asterisks,FIG. 2H), and, thus, their backbone conformations are not free to varyunless metal ions are lost, which is not consistent with ligand binding.Switching SDL3 to β₃-like with an Ala to Arg substitution abolishedpro-TGF-β1 binding and activation, while the Ala to Tyr substitution inSDL1 led to partial loss of binding and activation.

As the outermost and only non-metal binding SDL, the conformation ofSDL2 is free to vary in evolution, as shown herein in a comparisonbetween integrins with identical α-subunits, α_(V)β₃ and α_(V)β₆.Differences stem from presence of a cis-Pro in SDL2 of β₃ and packinginteractions with X₂ residues in SDL1 and SDL3. Consistent with the lackof effect shown herein of two SDL2 mutations, only one of six residuesbetween the two cysteines in SDL2 of β₆ and β₈ are identical (FIG. 2H),yet α_(V)β₆ and α_(V)β₈ bind and activate pro-TGF-β comparably well(FIG. 1A and FIG. 1B). This indicates backbone-dependent contributionsof SDL2 to integrin specificity.

As integrins are important therapeutic targets, the identification ofthe three SDLs of integrin β-subunits not only advances theunderstanding of how β-subunits contribute to integrin-ligandspecificity, but also the ability to rationally design antagonists.

Exemplary Material and Methods Used in Example 4

Pro-TGF-β cell surface binding and activation. α_(V) in a modified pEF1vector and β₁, β₃, β₅, β₆, or β₈ mutants in pcDNA3.1 (−) vector weretransiently transfected into 293T cells as described in Ref 11. Purifiedpro-TGF-β1 (17) was fluorescently labeled with fluoresceinisothiocyanate (FITC) using the Pierce (Thermo Fisher Scientific,Rockford, Ill.) FITC labeling kit according to manufacturer'sinstructions. Cells were resuspended in HBS buffer (20 mM HEPES, pH 7.4,137 mM NaCl, and 5 mM KCl, 5.5 mM glucose, and 1% bovine serum albumin)and incubated at room temperature for 30 min with 50 nM FITC-pro-TGF-β1in the presence of either (1) 5 mM EDTA, (2) 1 mM Mg²⁺/Ca²⁺, or (3) 1 mMMn²⁺/0.2 mM Ca²⁺ and subjected to flow cytometry without washing. Todetermine the expression of different α_(V) integrins, cells wereresuspended and incubated at room temperature for 30 min with 2 μg/mlP2W7 antibody (anti α_(V), Sigma-Aldrich, St. Louis, Mo.) and thenstained on ice for 30 min with FITC-anti-mouse IgG (1/500)(Sigma-Aldrich). Cells were washed once and subjected to fluorescentflow cytometry. Ligand binding was measured as the mean fluorescenceintensity (MFI) of pro-TGF-β1 after subtraction of the MFI in presenceof EDTA.

TGF-β assays used 293T cells transiently transfected with the α_(V) andβ subunits as above along with wild-type or mutant human pro-TGF-β1 inpcDNA3.1 (−), then co-cultured with transformed mink lung cellsexpressing a luciferase gene under the control of a TGF-β1-induciblepromoter (17, 21).

α_(V)β₆ and α_(V)β₃ headpiece expression and purification. Solubleα_(V)β₆ headpiece was prepared as follows. The α_(V) headpiece (residues1-594) with M400C mutation was followed by a 3C protease site, the ACIDcoiled-coil, a Strep-II tag and a His tag. β₆ headpiece residues 1-474with I270C or β₃ headpiece residues 1-472 with Q267C were followed bythe 3C site, the BASE coiled-coil, and a His tag. The Cys mutationsgenerated a disulfide bond that prevented α/β subunit dissociation.Proteins expressed in HEK293s Gnt I⁻ cells with Ex-Cell 293 serum freemedia (Sigma) were purified using Ni-NTA affinity column (Qiagen).Protein was cleaved by 3C protease at 4° C. overnight, passed throughNi-NTA resin and further purified using an ion exchange gradient from 50mM NaCl to 1M NaCl, 20 mM Tris-HCl pH 8.0 (Q fast-flow Sepharose, GEhealthcare) and gel filtration (Superdex 200, GE healthcare).

Fluorescence anisotropy. Fluorescence anisotropy was in 150 mM NaCl, 1mM Mg²⁺/Ca²⁺, 20 mM HEPES, pH 7.4 or buffer at indicated pH with 5 nMfluorescence probe (FITC-pro-TGF-β3 peptide, FITC-GRGDLGRL (SEQ ID NO:68)). Binding affinities were calculated as described in Ref 23. Insaturation binding assays, the anisotropy of the fluorescence probe wasmeasured while α_(V)β₆ headpiece (starting at 2.67 μM) or α_(V)β₃headpiece (starting at 75 μM) was serially diluted in 3-fold decrements.Competition binding assays used 200 nM α_(V)β₆ or 4 μM α_(V)β₃headpiece, 5 nM of fluorescence probe, and competing peptide seriallydiluted in 3-fold decrement from 500 μM to 0.5 nM.

α_(V)β₆ headpiece crystallization, data collection and structuredetermination. α_(V)β₆ crystals in hanging drops were formed with 3mg/ml α_(V)β₆ headpiece in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mMCaCl₂ and 1 mM MgCl₂ buffer (1 μl) and 1 μl reservoir solution of 20%PEG 4000, 0.1 M sodium cacodylate pH 6.0, 0.2 M ammonium sulfate.Identical vol:vol mixture of the protein and crystallization buffersyields a pH of 6.5. Pro-TGF-β3 peptide (1 mM each Ac-HGRGDLGRLKK-NH₂(SEQ ID NO: 78), MgCl₂, and CaCl₂, 0.2 μl) was added to drops of ˜1.5 μl(˜130 μM final conc.) for 4 h before harvesting crystals. Immediatelyprior to flash freezing in liquid N₂, crystals were dipped in reservoirsolution containing 25% glycerol with or without peptide, MgCl₂, andCaCl₂ each at 0.25 mM.

Diffraction data from GM/CA-CAT beamline 23-ID of Advanced Photon Source(APS) at Argonne National Laboratory were processed using XDS (24) withcross-correlation to determine the diffraction limit (25). Structureswere solved using molecular replacement by PHASER (26) with the α_(V)β₃headpiece from PDB code 4G1E as the search model (14). The structure wasrefined with PHENIX (27), manually built using Coot, and validated withMolprobity (28). I/σ(I) and CC½ in the highest resolution shell increaseas a function of the number of diffraction images in plots generatedwith XDS. Furthermore, Rwork/Rfree of the outer shell are 38.0%/41.7%and 39.9%/44.4% for the apo and peptide soaked-models respectively(Table 1). These results show that the weak diffraction data in theouter shell contribute to structure determination.

Example 5. Disulfide Linked α_(V)β₃ and α_(V)β₈ Integrin HeadpieceDimers

To generate the disulfide linked α_(V)β₃ and α_(V)β₈ integrin headpiecedimers, any one or more of the cysteine mutations in the α_(V) subunitas described herein can be used. In this example, amino acid residues399-401 (Ser-Met-Pro) of SEQ ID NO: 1 was substituted withSer-Gly-Cys-Pro (SEQ ID NO: 59) to introduce a cysteine in the α_(V)subunit. Cysteines were introduced in the β₃ subunit and the β₈ subunitin the same position structurally as in the β₆ subunit. That is, aminoacid residue 293 (Gln) of SEQ ID NO: 5 was mutated to Cys in the β₃subunit (SEQ ID NO: 5 includes the signal peptide sequence at positions1-26. Without the signal peptide sequence, the numbering of the aminoacid residue Gln being substituted with Cys is counted as amino acidresidue 267), and amino acid residue 301 (Val) of SEQ ID NO: 6 wasmutated to Cys in the β₈ subunit (SEQ ID NO: 6 includes the signalpeptide sequence at positions 1-42. Without the signal peptide sequence,the numbering of the amino acid residue Val being substituted with Cysis counted as amino acid residue 259). Stable cell lines secretingintegrin heterodimers were produced using a similar protocol forexpressing α_(V) β₆ as described in Example 4 above, and the heterodimermaterial was purified in good quantities. The modified α_(V)β₃ andα_(V)β₈ integrin headpiece dimers were formed with disulfide bond(s)cross-linking the α and β subunits, as in α_(V) β₆, as confirmed byreducing and non-reducing SDS page (FIG. 5). Without wishing to be boundby the theory, as the α and β subunits are crosslinked to each other byat least one or more disulfide bonds to form a heterodimer, thedisulfide linked integrin heterodimer generally has a better yield thanthe wildtype integrin, which is non-covalently linked and is thusreversible.

Example 6. Disulfide Linked 3-Domain Integrin Headpiece Fragment

Generally, a wild-type integrin α headpiece polypeptide includesβ-propeller domain and a thigh domain, while a wild-type integrin βheadpiece polypeptide generally includes a βI domain, a hybrid domain, aPSI (plexin, semaphoring, and integrin) domain, and an I-EGF-1 domain.Here, the inventors have surprisingly discovered that a 3-domainintegrin fragment of the α_(V)β₈ headpiece dimer, which contains onlythe α_(V) β-propeller and thigh domains and the β₆ βI domain, and iscrosslinked using a disulfide bond as described herein, can be generatedwith good expression. Such an integrin fragment has never beenpreviously made in good yield, but the inventors was successfully ableto express such a 3-domain integrin fragment by introducing a disulfidebond to crosslink the α headpiece fragment and the β headpiece fragment(FIG. 6). In one embodiment, the α_(V) headpiece has an amino acidsequence of SEQ ID NO: 1 with a M400C mutation and the β₆ βI domain hasan I270C mutation (the numbering is based on SEQ ID NO: 2). The aminoacid sequence of the β₆ βI domain is shaded in black as shown in FIG. 8(starting from DYP . . . ELR of SEQ ID NO: 2). The binding affinity ofthe 3-domain α_(V)β₆ integrin fragment to pro-TGF-β1 was measured, e.g.,by Biacore, and then compared to that of the corresponding fullheadpiece. The K_(d) of 3-domain α_(V)β₆ integrin fragment is 0.18±0.02nM, while the K_(d) of the corresponding full headpiece is 8.2±2.0 nM.

Example 7. Improved Crystal Resolution of Modified α_(V)β₃, α_(V)β₆, andα_(V)β₈ Integrin Dimers

With the disulfide bond modified integrin dimers as described in theprevious Examples, the resolution of integrin α_(V)β₃, α_(V)β₆, andα_(V)β₈ crystals have been greatly improved. The crystal structure ofα_(V)β₃ has been previously shown only at a resolution of around 3 Å orhigher. However, the inventors were able to obtain a higher-resolutioncrystal structure of α_(V)β₃, α_(V)β₆, and α_(V)β₈ using the modifiedintegrin headpiece with one or more disulfide bonds. With the disulfidelinked α_(V)β₃ integrin dimer, a resolution as high as about 1.9 Å waseven obtained. Data on representative integrin crystal structures areshown in Table 6.

TABLE 6 Statistincs of X-ray diffraction and structure refinement αvβ3αvβ6 αvβ8 Data collection Space Group P22₁2₁ C2 P1 α, β, γ ° 90, 90, 9090, 90, 98.7 90. 90. 110.0 Unit Cell (a, b, c) Å 87.1, 124.1, 165.3184.4, 170.0, 102.4 153.1, 55.3, 181.9 Resolution range (Å) 50.0-1.90(1.95-1.90) 50.0-2.50 (2.67-2.50) 50.0-2.90(2.98-2.90) Compeleteness (%)95.0 (95.9) 94.4 (70.6) 94.6 (88.6) Number reflections 259,318 (19,402)87,425 (5, 641) 118,116 (8,128) Redundancy 1.8 (1.8) 2.3 (2.1) 1.9(1.9)R_(merge) (%) ^(b) 11.3 (149) 11.6 (233) 18.6 (288) I/σ(I) 6.5 (0.57)4.9 (0.35) 4.4 (0.27) CC_(1/2) (%)^(c) 99.6 (17.2) 98.3 (10.4) 97.8(13.6) Wavelength (Å) 1.0332 1.0332 1.0332 Refinement R_(work) (%)^(d)19.2 23.6) R_(free) (%) 22.4 27.9 Bond RMSD (Å) 0.009 0.005 Angle RMSD(°) 1.13 0.76 Ramachandran plot^(c) 96.5/3.5/0.0 95.7/4.1/0.2(Favored/allowed/outlier) ^(a)The numbers in parentheses refer to thehighest resolution shell. ^(b) Rmerge = Σh Σi |Ii(h) − <I(h)> |/ΣhΣiIi(h), where Ii(h) and <I(h)> are the i^(th) and mean measurement of theintensity of reflection h. ^(c)Pearson's correlation coefficient betweenaverage intensities of random half-datasets for unique reflection.^(d)Rfactor = Σh||Fobs (h)|−|Fcalc (h)||/Σh|Fobs (h)|, where Fobs (h)and F calc (h) are the observed and calculated structure factors,respectively. No I/σ(I) cutoff was applied. ^(e)Calculated withMolProbity.

Example 8. Example Use of the Modified Integrin Polypeptide Dimers toScreen for Novel Inhibitory Drugs

Competition binding assays combined with fluorescence anisotropy assayswere performed to determine binding affinity of each candidate inhibitorto the appropriate integrin. Competition binding assays used 200 nMα_(V)β₆ or 300 nM α_(V)β₈ headpiece, 5 nM of fluorescence probe (thesame probe as used in Example 4, e.g., FITC-pro-TGF-β3 peptide orFITC-GRGDLGRL) as a competing peptide, and a candidate inhibitorserially diluted from 200 nM to 0.1 nM. FIGS. 7A-7B show fluorescenceanisotropy data of each indicated test indicator. Binding affinities ofeach test inhibitor were calculated as described in Rossi et al. (2011)Nat. Protoc. 6: 365, and shown in Table 7 below. The smaller the K_(d)value is, the stronger the binding affinity of the test inhibitor to thetarget integrin, and thus the more potent its inhibitory effect is.

TABLE 7 Computed K_(d) values of each indicated inhibitor for themodified α_(v)β₆ and α_(v)β₈ integrin polypeptide dimers describedherein Inhibi- Inhibi- Inhibi- Inhibi- Inhibi- Inhibi- (nM) tor 0 tor 3tor 7 tor 9 tor 11 tor 15 αvβ6 2.6  2.6 89.8 37.5  8.1  9.3 αvβ8 7.637.5 124 113 66.9 54.7

REFERENCES

All references cited herein, in the specification and Examples areincorporated in their entirety by

REFERENCE

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SEQUENCE LISTING (SEQ ID NO: 1)FNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSGCPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDTGG(SEQ ID NO: 2)HVQGGCALGGAETCEDCLLIGPQCAWCAQENFTHPSGVGERCDTPANLLAKGCQLNFIENPVSQVEILKNKPLSVGRQKNSSDIVQIAPQSLILKLRPGGAQTLQVHVRQTEDYPVDLYYLMDLSASMDDDLNTIKELGSRLSKEMSKLTSNFRLGFGSFVEKPVSPFVKTTPEEIANPCSSIPYFCLPTFGFKHILPLTNDAERFNEIVKNQKISANIDTPEGGFDAIMQAAVCKEKIGWRNDSLHLLVFVSDADSHFGMDSKLAGIVCPNDGLCHLDSKNEYSMSTVLEYPTIGQLIDKLVQNNVLLIFAVTQEQVHLYENYAKLIPGATVGLLQKDSGNILQLIISAYEELRSEVELEVLGDTEGLNLSFTAICNNGTLFQHQKKCSHMKVGDTASFSVTVNIPHCERRSRHIIIKPVGLGDALELLVSPECNCDCQKEVEVNSSKCHHGNGSFQCGVCACHPGHMGPRCESRG (SEQ ID ND: 3) 1

61 GFSVEFYRPG TDGVSVLVGA PKANTSQPGV LQGGAVYLCP WGASPTQCTP IEFDSKGSRL 121LESSLSSSEG EEPVEYKSLQ WFGATVRAHG SSILACAPLY SWRTEKEPLS DPVGTCYLST 181DNFTRILEYA PCRSDFSWAA GQGYCQGGFS AEFTKTGRVV LGGPGSYFWQ GQILSATQEQ 241IAESYYPEYL INLVQGQLQT RQASSIYDDS YLGYSVAVGE FSGDDTEDFV AGVPKGNLTY 301GYVTILNGSD IRSLYNFSGE QMASYFGYAV AATDVNGDGL DDLLVGAPLL MDRTPDGRPQ 361EVGRVYVYLQ HPAGIEPTPT LTLTGHDEFG RFGSSLTPLG DLDQDGYNDV AIGAPFGGET 421QQGVVFVFPG GPGGLGSKPS QVLQPLWAAS H T PDFFGSAL RGGRDLDGNG YPDLIVGSFG 481VDKAVVYRGR PIVSASASLT IFPAMFNPEE RSCSLEGNPV ACINLSFCLN ASGKHVADSI 541GFTVELQLDW QKQKGGVRRA LFLASRQATL TQTLLIQNGA REDCREMKIY LRNESEFRDK 601LSPIHIALNF SLDPQAPVDS HGLRPALHYQ SKSRIEDKAQ ILLDCGEDNI CVPDLQLEVF 661GEQNHVYLGD KNALNLTFHA QNVGEGGAYE AELRVTAPPE AEYSGLVRHP GNFSSLSCDY 721FAVNQSRLLV CDLGNPMKAG ASLWGGLRFT VPHLRDTKKT IQFDFQILSK NLNNSQSDVV 781SFRLSVEAQA QVTLNGVSKP EAVLFPVSDW HPRDQPQKEE DLGPAVHHVY ELINQGPSSI 841SQGVLELSCP QALEGQQLLY VTRVTGLNCT TNHPINPKGL ELDPEGSLHH QQKREAPSRS 901SASSGPQILK CPEAECFRLR CELGPLHQQE SQSLQLHFRV WAKTFLQREH QPFSLQCEAV 961YKALKMPYRI LPRQLPQKER QVATAVQWTK AEGSYGVPLW IIILAILFGL LLLGLLIYIL 1021YKLGFFKRSL PYGTAMEKAQ LKPPATSDA (SEQ ID NO: 4) 1

61 sarcddleal kkkgcppddi enprgskdik knknvtnrsk gtaeklkped itqiqpqqlv 121lrlrsgepqt ftlkfkraed ypidlyylmd lsysmkddle nvkslgtdlm nemrritsdf 181rigfgsfvek tvmpyisttp aklrnpctse qnctspfsyk nvlsltnkge vfnelvgkqr 241isgnldspeg gfdaimqvav cgsligwrnv trllvfstda gfhfagdgkl ggiv l pndgq 301chlennmytm shyydypsia hlvqklsenn iqtifavtee fqpvykelkn lipksavgtl 361sanssnviql iidaynslss evilengkls egvtisyksy ckngvngtge ngrkcsnisi 421gdevqfeisi tsnkcpkkds dsfkirplgf teevevilqy icececqseg ipespkcheg 481ngtfecgacr cnegrvgrhc ecstdevnse dmdaycrken sseicsnnge cvcgqcvcrk 541rdntneiysg kfcecdnfnc drsnglicgg ngvckcrvce cnpnytgsac dcsldtstce 601asngqicngr gicecgvckc tdpkfqgqtc emcgtclgvc aehkecvqcr afnkgekkdt 661ctqecsyfni tkvesrdklp qpvqpdpvsh ckekdvddcw fyftysvngn nevmvhvven 721pecptgpdii pivagvvagi vliglallli wkllmiihdr refakfekek mnakwdtgen 781piyksavttv vnpkyegk (SEQ ID NO: 5) 1

61 sprcdlkenl lkdncapesi efpvsearvl edrplsdkgs gdssqvtqvs pqrialrlrp 121ddsknfsiqv rqvedypvdi yylmdlsysm kddlwsignl gtklatqmrk ltsnlrigfg 181afvdkpvspy myisppeale npcydmkttc lpmfgykhvl tltdqvtrfn eevkkqsysr 241nrdapeggfd aimqatvcde kigwrndash llvfttdakt hialdgrlag iv q pndgqch 301vgsdnhysas ttmdypslgl mteklsqkni nlifavtenv vnlyqnysel ipgttvgvls 361mdssnvlgli vdaygkirsk velevrdlpe elslsfnatc lnnevipglk scmglkigdt 421vsfsieakvr gcpqekeksf tikpvgfkds livqvtfdcd cacqaqaepn shrcnngngt 481fecgvcrcgp gwlgsqcecs eedyrpsqqd ecspregqpv csgrgeclcg qcvchssdfg 541kitgkycecd dfscvrykge mcsghgqcsc gdclcdsdwt gyycncttrt dtcmssngll 601csgrgkcecg scvciqpgsy gdtcekcptc pdactfkkec veckkfdrga lhdentcnry 661crdeiesvke lkdtgkdavn ctykneddcv vrfqyyedss gksilyvvee pecpkgpdil 721vvllsvmgai lliglaalli wkllitihdr kefakfeeer arakwdtann plykeatstf 781tnityrgt (SEQ ID NO: 6) 1

61 lgpecgwcvq edfisggsrs ercdivsnli skgcsvdsie ypsvhviipt eneintqvtp 121gevsiqlrpg aeanfmlkvh plkkypvdly ylvdvsasmh nnieklnsvg ndlsrkmaff 181srdfrlgfgs yvdktvspyi sihperihnq csdynldcmp phgyihvlsl tenitefeka 241vhrqkisgni dtpeggfdam lqaavceshi gwrkeakrll lvmtdqtshl aldsklagiv 301 vpndgnchlk nnvyvksttm ehpslgqlse klidnninvi favqgkqfhw ykdllpllpg 361tiageieska anlnnlvvea yqklisevkv qvenqvqgiy fnitaicpdg srkpgmegcr 421nvtsndevlf nvtvtmkkcd vtggknyaii kpigfnetak ihihrncscq cednrgpkgk 481cvdetfldsk cfqcdenkch fdedqfsses ckshkdqpvc sgrgvcvcgk cschkiklgk 541vygkycekdd fscpyhhgnl caghgeceag rcqcfsgweg drcqcpsaaa qhcvnskgqv 601csgrgtcvcg rcectdprsi grfcehcptc ytackenwnc mqclhphnls qaildqckts 661calmeqqhyv dqtsecfssp sylriffiif ivtfligllk vliirqvilq wnsnkiksss 721dyrvsaskkd klilqsvctr avtyrrekpe eikmdiskln ahetfrcnfAn exemplary amino acid sequence of a modified integrin α_(v) headpiece polypeptide describedherein (comprising a β-propeller domain and a thigh domain) (upon 3C protease site cutting).The underlined is the substitution introduced to permit a disulfide bond.(SEQ ID NO: 131)FNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSGCPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDTGGLEVLFQAn exemplary amino acid sequence of a purified, modified integrin β₆ headpiece polypeptidedescribed herein (comprising (i) a βI domain without other domains (i.e., no hybrid domain, noPSI domain, and no EGF-1 domain) and (ii) a detectable label such as His₆ Tag (SEQ ID NO:132) in the sequence below). The His₆ Tag can be uncleavable. The underlined is thesubstitution introduced to permit a disulfide bond. (SEQ ID NO: 133)QTEDYPVDLYYLMDLSASMDDDLNTIKELGSRLSKEMSKLTSNFRLGFGSFVEKPVSPFVKTTPEEIANPCSSIPYFCLPTFGFKHILPLINDAERFNEIVKNQKISANIDTPEGGFDAIMQAAVCKEKIGWRNDSLHLLVFVSDADSHFGMDSKLAGIVCPNDGLCHLDSKNEYSMSTVLEYPTIGQLIDKLVQNNVLLIFAVTQEQVHLYENYAKLIPGATVGLLQKDSGNILQLIISAYEELRSEHHHHHHAn exemplary amino acid sequence of a purified, modified integrin β₈ headpiece polypeptidedescribed herein (comprising (i) a βI domain and a hybrid domain without other domains (i.e.,no PSI domain and no EGF-1 domain). The underlined is the substitution introduced to permit adisulfide bond. (SEQ ID NO: 134)SKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIYFNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSSRGLQTLFQAn exemplary amino acid sequence of a purified, modified integrin β₆ headpiece polypeptidedescribed herein (comprising a full headpiece including a βI domain, a hybrid domain, a PSIdomain, and an EGF-1 domain). (SEQ ID NO: 135)HVQGGCALGGAETCEDCLLIGPQCAWCAQENFTHPSGVGERCDTPANLLAKGCQLNFIENPVSQVEILKNKPLSVGRQKNSSDIVQIAPQSLILKLRPGGAQTLQVHVRQTEDYPVDLYYLMDLSASMDDDLNTIKELGSRLSKEMSKLTSNFRLGFGSFVEKPVSPFVKTTPEEIANPCSSIPYFCLPTFGFKHILPLTNDAERFNEIVKNQKISANIDTPEGGFDAIMQAAVCKEKIGWRNDSLHLLVFVSDADSHFGMDSKLAGIVCPNDGLCHLDSKNEYSMSTVLEYPTIGQLIDKLVQNNVLLIFAVTQEQVHLYENYAKLIPGATVGLLQKDSGNILQLIISAYEELRSEVELEVLGDTEGLNLSFTAICNNGTLFQHQKKCSHMKVGDTASFSVTVNIPHCERRSRHIIIKPVGLGDALELLVSPECNCDCQKEVEVNSSKCHHGNGSFQCGVCACHPGHMGPRCESRGLQTLFQAn exemplary amino acid sequence of a purified, modified integrin β₃ headpiece polypeptidedescribed herein (comprising a full headpiece including a βI domain, a hybrid domain, a PSIdomain, and an EGF-1 domain). (SEQ ID NO: 136)GPNICTTRGVSSCQQCLAVSPMCAWCSDEALPLGSPRCDLKENLLKDNCAPESIEFPVSEARVLEDRPLSDKGSGDSSQVTQVSPQRIALRLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSYSMKDDLWSIQNLGTKLATQMRKLTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVLTLTDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIGWRNDASHLLVFTTDAKTHIALDGRLAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEKLSQKNINLIFAVTENVVNLYQNYSELIPGTTVGVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVIPGLKSCMGLKIGDTVSFSIEAKVRGCPQEKEKSFTIKPVGFKDSLIVQVTFDCDCACQAQAEPNSHRCNNGNGTFECGVCRCGPGWLGSQCESRGLQTLFQAn exemplary amino acid sequence of a purified, modified integrin β₈ headpiece polypeptidedescribed herein (comprising a full headpiece including a βI domain, a hybrid domain, a PSIdomain, and an EGF-1 domain). (SEQ ID NO: 137)EDNRCASSNAASCARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIYFNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKSRGLQTLFQ

1-78. (canceled)
 79. A modified integrin polypeptide dimer, comprisinga. a modified integrin alpha-v headpiece polypeptide comprising an aminoacid sequence with at least one cysteine residue mutation introduced inthe alpha-v headpiece polypeptide; and b. a modified integrin beta-8headpiece polypeptide with at least one cysteine residue introducedthereto, wherein the modified integrin alpha-v headpiece polypeptide andthe modified integrin beta-8 headpiece are covalently linked by at leastone disulfide bond.
 80. The modified integrin polypeptide dimer of claim79, wherein the modified integrin alpha-v headpiece polypeptidecomprises an amino acid sequence with at least one cysteine residuemutation introduced in the beta-propeller domain of the alpha v subunitof the alpha-v headpiece polypeptide.
 81. The modified integrinpolypeptide dimer of claim 80, wherein the modified integrin beta-8headpiece comprises one or more domains selected from the groupconsisting of a PSI domain, hybrid domain, beta-I domain and an EGF-1domain.
 82. The modified integrin polypeptide dimer of claim 79, whereinthe modified integrin beta-8 headpiece comprises one or more domainsselected from the group consisting of a PSI domain, hybrid domain,beta-I domain and an EGF-1 domain.
 83. The modified integrin polypeptidedimer of claim 79, wherein the modified integrin beta-8 headpiececomprises the PSI domain, the hybrid domain, and the beta-I domain. 84.The modified integrin polypeptide dimer of claim 79, wherein themodified integrin alpha-v headpiece polypeptide consists essentially ofthe beta-propeller domain and the thigh domain.
 85. The modifiedintegrin polypeptide dimer of claim 79, wherein the modified integrinbeta headpiece comprises a modified integrin beta-8 headpiecepolypeptide comprising residues 43 to 498 of SEQ ID NO:6 with at leastone cysteine residue introduced thereto.
 86. The modified integrinpolypeptide dimer of claim 79, wherein the modified integrin betaheadpiece is a modified integrin beta-8 headpiece polypeptide of SEQ IDNO:6 with at least one cysteine residue introduced thereto.
 87. Themodified integrin polypeptide dimer of claim 79, wherein the at leastone cysteine residue introduced to the polypeptide of SEQ ID NO:6comprises substitution of amino acid residue 301 (Val) with Cys.
 88. Themodified integrin polypeptide dimer of claim 79, wherein the modifiedintegrin beta-8 headpiece polypeptide does not include a signal peptidesequence.
 89. The modified integrin polypeptide dimer of claim 79,comprising an amino acid sequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu (SEQID NO: 66), wherein X₁, X₂, and X₃ are each independently an amino acidmolecule.
 90. A modified integrin dimer comprising a modified integrinalpha-v headpiece polypeptide comprising an amino acid sequence with atleast one cysteine residue mutation introduced in alpha-v headpiecepolypeptide, the modified integrin alpha-v headpiece polypeptidecomprising at least one cysteine residue mutation introduced in thealpha-v headpiece polypeptide, covalently bound to a modified integrinbeta-8 headpiece polypeptide comprising at least one cysteine residueintroduced thereto by at least one disulfide bond.
 91. The modifiedintegrin dimer of claim 90, wherein the modified integrin beta-8headpiece polypeptide comprises residues 43 to 498 of SEQ ID NO:6 withat least one cysteine residue introduced thereto.
 92. The modifiedintegrin dimer of claim 91, wherein the modified integrin beta-8headpiece polypeptide does not comprise a signal peptide sequence. 93.The modified integrin dimer of claim 90, wherein the modified integrinbeta-8 headpiece polypeptide does not comprise a signal peptidesequence.
 94. The modified integrin dimer of claim 90, wherein themodified integrin beta-8 headpiece polypeptide comprises SEQ ID NO:6with at least one cysteine residue introduced thereto.
 95. The modifiedintegrin dimer of claim 94, wherein the modified integrin beta-8headpiece polypeptide comprises substitution of amino acid residue 301(Val) of SEQ ID NO:6 with Cys.
 96. The modified integrin dimer of claim90, wherein the modified integrin dimer comprises comprising an aminoacid sequence of X₃-Arg-Gly-Asp-Leu-X₁-X₂-Leu (SEQ ID NO: 66), whereinX₁, X₂, and X₃ are each independently an amino acid molecule.
 97. Amodified integrin beta-8 headpiece polypeptide comprising amino acidresidues 43 to 498 of SEQ ID NO: 6 with at least one Cys residueintroduced thereto by substitution of amino acid residue 301 (Val) withCys.
 98. The modified integrin beta-8 headpiece polypeptide of claim 97,wherein the modified integrin beta-8 headpiece polypeptide comprisessubstitution of amino acid residue 301 (Val) of SEQ ID NO:6 with Cys.